Pathogenesis of Breast Cancer

#### **Chapter 7**

## Regulation of Exosomes in the Pathogenesis of Breast Cancer

*Congjian Shi, Hongqin Yang, Zhengchao Wang and Zhenghong Zhang*

#### **Abstract**

Extracellular vesicles (EVs) are a heterogeneous group of endogenous nanoscale vesicles that are secreted by various cell types. Based on their biogenesis and size distribution, EVs can be broadly classified as exosomes and microvesicles. Exosomes are enveloped by lipid bilayers with a size of 30–150 nm in diameter, which contain diverse biomolecules, including lipids, proteins and nucleic acids. Exosomes transport their bioactive cargoes from original cells to recipient cells, thus play crucial roles in mediating intercellular communication. Breast cancer is the most common malignancy among women and remains a major health problem worldwide, diagnostic strategies and therapies aimed at breast cancer are still limited. Growing evidence shows that exosomes are involved in the pathogenesis of breast cancer, including tumorigenesis, invasion and metastasis. Here, we provide a straightforward overview of exosomes and highlight the role of exosomes in the pathogenesis of breast cancer, moreover, we discuss the potential application of exosomes as biomarkers and therapeutic tools in breast cancer diagnostics and therapeutics.

**Keywords:** extracellular vesicles, exosomes, breast cancer, miRNAs

#### **1. Introduction**

Extracellular vesicles (EVs) are heterogeneous membrane-bound vesicles which originate from endosomal or plasma membrane called exosomes or microvesicles, respectively [1]. The release of EVs was initially identified as a mode for cells to eliminate unwanted substances, however, the initial view with regard to EVs has changed dramatically with the deepening of research, and their crucial roles in diverse physiological and pathological processes have attracted extensive attention. According to their original cells, EVs are loaded with a specific set of preassembled bioactive cargoes, and give rise to phenotypic and genotypic changes in recipient cells [2, 3]. These cargoes enclosed within EVs are biologically significant, for example, three EV subtypes including one microvesicle and two exosome populations released by LIM1863 CRC (colorectal cancer) cells have distinct miRNA expression profiles [4]. EVs contribute to numerous aspects of normal physiological processes, including blood coagulation, immune surveillance, tissue repair and stem cell maintenance [5]. They are also closely related with diverse human diseases, including cancer, infectious diseases, neurologic diseases and cardiometabolic diseases [6]. Exosomes are a subtype of EVs and the application of exosomes as biomarkers

and therapeutic tools has appeared as a promising area of research due to some preponderant properties of exosomes. Exosomes can be released according to the command received from adjacent and distant cells, or in response to the stimulation induced by local conditions [7]. Both normal and pathological cells are capable of secreting exosomes and they are stable in biological fluids [8]. Breast cancer is the most common malignancy affecting women, and its morbidity and mortality are estimated to increase in the coming years [9]. One in eight to ten women will be diagnosed with breast cancer during their lifetime [10], and breast cancer has seriously affected women's health. Accumulating evidence indicates that exosomes are involved in the pathogenesis of breast cancer, including tumorigenesis, invasion and metastasis. Studies focused on exosomes might provide novel perspectives for revealing breast cancer pathogenesis and improving the current poor diagnostic and therapeutic status of breast cancer.

#### **2. Extracellular vesicles**

Cells naturally release EVs into the extracellular space, these nanoscale vesicles encompassing bioactive cargoes play crucial roles in diverse physiological and pathological processes. The term EVs represent several subtypes of vesicles, standardized criteria for distinguishing EVs subtypes are still under discussion, but it is universally acknowledged that they can be classified as two main categories: exosomes and microvesicles. Other EVs subtypes such as apoptotic bodies [11], spheresomes [12] and large oncosomes [13], are not mentioned in this review. Exosomes have endosomal origin, they are 30–150 nm in diameter and float at a density of 1.13–1.19 g/ml in sucrose gradient [14, 15]. Exosomes are essentially intraluminal vesicles (ILVs) generated by inward budding of endosomal membrane during the maturation of endosomes, then released to the extracellular space when multivesicular bodies (MVBs) (also referred to late endosomes) fuse with plasma membranes [16, 17]. Microvesicles, typically larger than exosomes (100–1000 nm in diameter), arise through direct outward budding and fission of plasma membrane [18], hence, the membrane composition of microvesicles can better reflect the membrane composition of original cells in contrast to exosomes. Although the origin of exosomes and microvesicles occurs at distinct intracellular locations, some common mechanisms participate in both processes. The modes by which recipient cells take up EVs including endocytosis, direct membrane fusion and receptor ligand binding [19], but the specific molecular mechanisms deserve further investigation.

At present, the biogenesis of EVs, the substances they contain and the biological effects they promote have been extensively studied, which make people find out the potential of EVs in clinical application. EVs subtypes like exosomes and microvesicles may perform different functions, and it is absolutely necessary to isolate high-purity EVs subtypes, which will be crucial for EV-related functionality and therapeutic value studies. But even in EV preparations with high-purity, electron microscopy (EM) results imply that they still contain co-purifying elements [20]. The isolation of EVs is challenging because EVs subtypes have some similarities, including their size, density, composition, and surface marker proteins [21]. Meanwhile, EVs derived from biological fluids contain a mixture of multiple EVs secreted by various cell types [22]. Therefore, it is imperative to formulate universal standard protocols for the preparation of EVs.

Due to some peculiar characteristics of EVs, they have prominent biotechnological potential. EVs are biocompatible and safe, coupled with nanoscale diameter, resulting in their long blood circulation half-life and high drug loading capacity, which makes them possible to be ideal drug delivery vehicles [23]. EVs represent

*Regulation of Exosomes in the Pathogenesis of Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.95858*

an attractive group of therapeutic biomarkers and has tremendous potential in immune response regulation and tissue regeneration [5, 24]. EVs are extensively found in diverse bodily fluids, and it is a promising area to serve EVs as biomarkers for early diagnosis and accurate prognosis. Since EVs are derived from bodily fluids, the diagnostic methods are probably non-invasive and considerably less painful than some existing diagnostic methods (for example, liver biopsy). Meanwhile, the clinical application of EVs can also monitor the response of therapy, which will contribute to convalescent process.

#### **3. Exosomes**

Exosomes are enveloped by lipid bilayers and act as mediators of intercellular communication through transmitting diverse functional biomolecules from original cells to recipient cells, and they are secreted by virtually all cell types, such as stem cells, immune cells and tumor cells [25]. The cargoes transported by exosomes including lipids, proteins, RNA (coding and non-coding) and even DNA (genomic and mitochondrial) [26]. Exosomes can be detected and isolated from diverse bodily fluids, exemplified by blood, urine, saliva, cerebrospinal fluid and breast milk [27], they can also be obtained from cell culture-conditioned media [28]. Some specific surface proteins are considered as the makers of exosome, such as tetraspanin family (CD9, CD63 and CD81), heat shock protein 70 (HSP70) and major histocompatibility complex (MHC) molecules [29]. Exosomes also contain abundant ceramide, cholesterol and sphingomyelin, which may relate to their lipid raft microdomains [30]. Multiple genetic materials are detected in exosomes, and exosome-encapsulated miRNAs have obtained extra attention because of their vital roles in regulating gene expression and can be used as biomarkers for a variety of diseases [31].

Exosomes exhibit unique biogenesis mechanism. Plasma membrane buds inward through endocytosis, resulting the generation of early endosomes [32]. The process from early endosomes to late endosomes (also referred to MVBs) requires the involvement of Golgi complex, during which ILVs also accumulate by the invagination of endosomal membrane in their lumen [15]. Then, MVBs either fuse with lysosomes, which ILVs are degraded, or fuse with plasma membranes, which ILVs are released to the extracellular space as exosomes [33]. Fusion of MVBs with plasma membrane requires the assistance of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) complexes [34]. The endosomal sorting complex required for transport (ESCRT) machinery, a vital participant in exosome biogenesis, is responsible for ILVs formation and protein sorting [35]. ESCRT machinery contains four complexes, ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III, as well as associated proteins, including ALIX, VPS4 and VTA1 [36]. ESCRT-0 recognizes the ubiquitinated cargoes, ESCRT-I and ESCRT-II initiate the budding process of ILVs, whereas ESCRT-III terminates this process [3, 37]. ESCRT-independent mechanisms also exist by the evidence that MVBs still form in the absence of ESCRTs [38], further studies report that the mechanisms are related with the sphingolipid ceramide [39] or some members of the tetraspanin family [40]. In single MVBs, a competitive relationship between ESCRT-dependent and ESCRT-independent mechanisms exists, which affects the size of ILVs formed inside [41], this makes it possible to identify different subpopulations of MVBs based on their ILVs size. A group of Rab GTPases including Rab11, Rab27a, and Rab27b, are also involved in the release of exosomes [42]. Once exosomes are released to the extracellular space, they may exist in the circulation or be taken up by adjacent and distant cells [43, 44].

Exosomal preparations with high-purity are significant for further exploration of exosomal biogenesis and functions, and techniques for exosomal isolation have made great advances. At present, the commonly used isolation techniques including ultracentrifugation-based techniques, size-based techniques, precipitation, immunocapture and microfluidic-based techniques [35, 45] (**Table 1**). Among them, ultracentrifugation is the most extensively used exosomal isolation technique for bodily fluids and cell culture supernatant [46]. Each technique has its own merits and demerits, and the combination of aforementioned techniques may lead to a more desirable isolation. Recent study showed that the acidic condition was more suitable for the isolation of exosomes [47], indicating that local pH of exosomes should be taken into account for future researches.

Specific roles of the tumor microenvironment during cancer progression and metastasis have been widely studied [48], and cancer cell–derived exosomes can establish a favorable microenvironment to induce cell proliferation, angiogenesis, resistance to apoptosis and initiation of pre-metastatic niches through their bioactive content [22, 49]. The secretion of exosome appears to have an impact on drug resistance, for example, exosomes enriched in TAG72 imply that CRC patients might be resistant to 5-FU [50]. And cells under pathological status release even more exosomes, it is estimated that there are approximately 2,000 trillion exosomes presented in normal human blood and 4,000 trillion exosomes presented in the blood of cancer patients [51]. According to these results, it is feasible to serve exosomes as biomarkers for diagnosis and prognosis. Exosomes are capable of inducing anti-tumor responses through delivering tumor antigens to immune cells, and exosomes derived from T cells can suppress tumor development [52], demonstrating their great potential in modulating immune responses. Enlightened by the capability of exosomes that transmits biomolecules from original cells to recipient cells, accompanied with their biocompatibility, low immunogenicity and toxicity, high stability in the circulation, biological barrier permeability and potential targeting to specific sites [53], diverse strategies have been developed for loading therapeutic cargoes into exosomes, which have a broad application prospect.

Exosome plays important roles in tumor diagnostics and therapeutics. Tissue biopsy is usually acquired from the site of primary tumor and reflects its molecular traits over a period of time, therapies will be determined according to the results of tissue biopsy. However, the limitations of tissue biopsy are obvious, it is not comprehensive enough to reflect heterogeneity and dynamic evolution of tumor [54].


#### **Table 1.**

*The advantages and disadvantages of different techniques used for exosome isolation.*

*Regulation of Exosomes in the Pathogenesis of Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.95858*

Exosome related liquid biopsy techniques including surface-enhanced Raman spectroscopy (SERS), next generation sequencing (NGS), digital droplet PCR (ddPCR) and molecular barcoding, have drawn extra attention due to its unique advantages of minimally invasive and serial biochemical tests [55]. Among diversified methods developed for liquid biopsy, SERS-based technique for detection of circulating tumor markers including exosomes is one of the most powerful methods, it owns the advantages of high sensitivity, specificity, tremendous spectral multiplexing capacity for simultaneous target detection, and its unique capability for obtaining intrinsic fingerprint spectra of biomolecules [56]. The application of exosome related liquid biopsy enables the improvement of various aspects of tumor management including early diagnosis and screening, prediction of prognosis, early detection of relapse, serial sampling and efficient longitudinal monitoring of disease progress and response to treatment [57]. Although exosome related liquid biopsy is a promising area, there are still some loopholes including difficult extraction and did not analyze the phenotypic studies of cells from tumor, that require further refinement and validation [58].

#### **4. Breast cancer**

Breast cancer is a disease with high heterogeneity, containing multiple tumor entities that have diverse clinical behavior and biological features [59], which complicate its diagnosis and treatment. Among women, breast cancer is the most common malignancy and the second leading causes of cancer-related death [60]. The 5-year overall survival rate for non-metastatic breast cancer patients is greater than 80%, whereas distant metastasis can reduce this rate to approximately 25%, and the main metastatic sites including bone, brain, liver and lung [61]. The diagnosis and treatment of patients are evaluated by clinical assessment, breast imaging, tumor size, histologic grade, lymph node involvement or acknowledged biomarkers, including estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2) and progesterone receptor (PR) [62]. The clinical classification of breast cancer should be reasonable, so as to select the most appropriate diagnostic strategy and therapy for each patient, and the molecular subtypes of breast cancer are represented by basal-like, HER2-enriched, normal breast-like, luminal A, luminal B and claudin-low [63]. The occurrence of breast cancer is influenced by age, race, obesity, smoke, drinking, oral contraceptives and other exogenous estrogens, age at menarche, age at menopause, age at first live birth and environmental toxins [64, 65], also, inherited genetic mutations are responsible for 5–10% of all breast cancer cases, and mutations in BRCA1 and BRCA2 are believed to increase the lifetime risk of being diagnosed with breast cancer by more than four times [66].

Breast cancer is generally diagnosed by mammography, computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, core needle biopsy, excisional biopsy and histopathologic evaluation [65, 67]. Diagnosed patients with parallel clinical and biological characteristics may exhibit distinct responses to treatment and bring about different outcomes [68], therefore, the research on breast cancer treatment needs to be further deepened. At present, surgery and radiation are mentioned frequently in treatment, and three approaches are primarily adopted in medical oncology: ER + -related breast cancer aimed at anti-endocrine strategies, HER2 + -related breast cancer treated with HER2-targeted drugs and triple-negative breast cancer (TNBC) managed with traditional cytotoxic therapy [69]. More importantly, it is not just about choosing the appropriate treatment for each patient, the sequencing of therapies should also be considered.

### **5. Exosomal functionality and therapeutic value in breast cancer**

It has been widely acknowledged that exosomes are important players in the pathogenesis of breast cancer (**Figure 1**). The release of exosomes induced by heparanase, hypoxia and other stimulation is involved in breast cancer angiogenesis, which facilitate tumorigenesis process of breast cancer [70]. Also, exosomes promote breast cancer tumorigenesis by modifying tumor microenvironment to permissive niches. Typically, alteration in miRNAs expression have been found to influence initiation and development of breast cancer [71], for example, compared with non-malignant breast cells or non-metastatic breast cancer cells, exosomal miR-10b is significantly upregulated in metastatic breast cancer cells [72]. Further research shows that RNA induced silencing complex-loading complex (RLC) proteins Dicer, AGO2 and TRBP, which have been proved to participate in miRNA biogenesis, can be detected in exosomes derived from the serum of breast cancer patients and breast cancer cells, moreover, Dicer inhibition in cancer exosomes obviously decelerates tumor growth in recipient cells [73]. Invasion plays an important role in cancer development, invasion ability of non-malignant breast cells can be induced by exosomes derived from metastatic breast cancer cells [72]. Metastatic breast cancer cells specifically express and release miR-105, during which exosomal miR-105 can transfer to endothelial cells and acts as an effective regulator of their migration [74]. Recent study also suggested that miR-7641 was identified as an important component of exosomes that could promote breast tumor metastasis [75]. Drug resistance are also closely related with exosomes as they are capable of transporting anti-cancer drugs outside breast cancer cells. Chen et al. reported that drug-resistant breast cancer cells might spread their drug-resistant capacity to sensitive cells through secreting exosomes, they further confirmed that this

#### **Figure 1.**

*Exosome in the pathogenesis of breast cancer. Exosome contribute to oncogenic transformation, angiogenesis, permissive niche formation and drug resistance in breast cancer.*

*Regulation of Exosomes in the Pathogenesis of Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.95858*

process was mediated by exosomal miRNAs [76]. Trastuzumab is a commonly used drugs to treat breast cancer, while exosome-transmitted cicHIPK3 could promote trastuzumab chemoresistance of drug-sensitive BC cells, decreasing the therapeutic effect [77]. Recent study showed that exosomal miR-155 regulated drug resistance of breast cancer [78] and chemotherapy with miR-155-targeting therapies may lead to satisfactory outcomes.

Breast cancer is a disease which tends to metastasis, patients with early diagnosis, reasonable prognosis and accurate treatment usually have more favorable outcomes, yet approaches against breast cancer are still limited, and exosomes could be employed as novel biomarkers and therapeutic tools for patients with breast cancer. Hannafon and colleagues found that exosomes derived from breast cancer cells were enriched with specific miRNAs (miR-1246 and miR-21), what's more, these miRNAs in plasma exosomes of breast cancer patients were significantly higher than those of healthy control subjects [79], and exosomes may play crucial roles as biomarkers for breast cancer. Distant metastasis or local recurrence of breast cancer are strongly related with exosomal miRNAs, including miR-17-5p, miR-93-5p, miR-130a-3p, miR-340-5p [80], which can serve as indicators for prognosis. Now that exosomes remain stable in biological fluids, they are also promising for early diagnosis or monitoring the treatment process of breast cancer. In contrast to delivering anticancer drugs outside breast cancer cells, exosomes can also target anticancer drugs to breast cancer cells after appropriate modifications, for example, exosomes modified by targeting ligands deliver doxorubicin to tumors [81], which improve the therapeutic efficacy. Exosomes derived from mesenchymal stem cells (MSCs) can be used as drug delivery vehicles to transport locked nucleic acid (LNA)-antimiR-142-3p, therefore reducing tumorigenicity in breast cancer [82].

#### **6. Summary**

Over the past few decades, on account of great advances in our understanding of breast cancer biology, diverse diagnostic and prognostic strategies, as well as targeted therapies are continuously evolving, while the situation of breast cancer patients remains unsatisfactory. For prevention and treatment of breast cancer, we need not only to develop new biomarkers and therapeutic tools, but also to further investigate the potential molecular mechanisms. Fortunately, accompany by our comprehension of exosomes is becoming more refined, the role of exosomes in initiation and development of breast cancer has been widely explored, and it is meaningful to translate exosomal research achievements to develop safe and effective therapies, diagnostic methods, along with drug delivery vehicles, which may conduce to improve the unsatisfactory situation of breast cancer patients.

#### **Acknowledgements**

This study was supported by the Special Funds of the Central Government Guiding Local Science and Technology Development (2020L3008), Provincial Natural Science Foundation (2018J01721, 2018J01721, 2019J01674 and 2020J01176), the Educational Reform Project (Y201809 and I202003009) and Training Program of Innovation and Entrepreneurship for Undergraduates (CXXL2020291 and CXXL2020293) of the Fujian Normal University.

*Global Women's Health*

### **Author details**

Congjian Shi, Hongqin Yang, Zhengchao Wang and Zhenghong Zhang\* Provincial Key Laboratory for Developmental Biology and Neurosciences, Key Laboratory of Optoelectronic Science and Technology for Medicine of Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China

\*Address all correspondence to: zhangzh@fjnu.edu.cn

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Regulation of Exosomes in the Pathogenesis of Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.95858*

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[66] J.A. de la Mare, L. Contu, M.C. Hunter, B. Moyo, J.N. Sterrenberg, K.C. Dhanani, L.Z. Mutsvunguma, A.L. Edkins, Breast cancer: current developments in molecular approaches to diagnosis and treatment, Recent Pat Anticancer Drug Discov 9(2) (2014) 153-175.

[67] E.S. McDonald, A.S. Clark, J. Tchou, P. Zhang, G.M. Freedman, Clinical Diagnosis and Management of Breast Cancer, J Nucl Med 57 Suppl 1 (2016) 9S–16S.

[68] E.A. Rakha, M.E. El-Sayed, J.S. Reis-Filho, I.O. Ellis, Expression profiling technology: its contribution to our understanding of breast cancer, Histopathology 52(1) (2008) 67-81.

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#### **Chapter 8**

### Long-Term Survivors of Breast Cancer: A Growing Population

*Nadeem Bilani, Elizabeth Blessing Elimimian, Leah Elson, Hong Liang and Zeina Nahleh*

#### **Abstract**

Breast cancer represents the most common malignancy among women. However, due to effective public health campaigns and updated screening guidelines, the annual incidence of late stage diagnoses has fallen. This stage migration has allowed for better prognosis and more women achieving long-term survival. In this chapter, we review long-term survivorship – defined as 10 years from diagnosis – as reported in the United States and around the world. Additionally, we provide analysis for socio-demographic, clinical and pathologic factors associated with 10-year survival, using data from a large national registry. This chapter also utilizes historical case data to forecast stage migration patterns in breast cancer diagnoses, within the United States, to 2030. Finally, we discuss the effects of the novel coronavirus pandemic on breast cancer treatment and access to care, with a review of clinical considerations for the future.

**Keywords:** breast cancer, epidemiology, survivors, clinical considerations, forecasting

#### **1. Introduction**

In 2015, the World Health Organization reported that cancer ranked within the top four reasons for death, before the age of 70, in 113 of the 172 countries surveyed [1]. The impact of cancer on women's health is incontrovertible. An estimated 2.1 million individuals around the world were diagnosed with breast cancer in 2018, alone [2]. It is the most common malignancy in women, matched only in Sub-Saharan Africa by cervical cancer, due to an elevated prevalence of tumorigenic strains of the human papillomavirus [3].

The breast cancer disease burden is expected to increase, due to a number of socioeconomic risk factors, including: aging and growth of the population, nulliparity, later maternal age at first pregnancy, the use of exogenous hormones (i.e. oral contraceptive pills, hormonal replacement therapy), alcohol intake, and obesity [4].

In addition to this rising incidence rate, outcomes in breast cancer are improving over time. In the United States, mortality has dropped by 40% between 1989 and 2017 [5]. This is thought to be due to a combination of a) mass screening campaigns that allow caregivers to diagnose the disease at earlier stages, thus offering

better prognoses, and b) the evolution of targeted and increasingly-efficacious therapeutics.

The relative indolence of most non-metastatic breast neoplasms, compared to other malignancies with more acute courses, makes reports of 5-year overall survival less clinically relevant, except in patients who already have limited life expectancy. Additionally, certain breast cancers may be associated with a high rate of late recurrence. For instance, patients with primary tumors that are estrogen receptor (ER)-positive develop distant metastasis in 10–20% of cases, five or more years following initial diagnosis [6]. Therefore, there is great utility in surveying literature, which reports *long-term* survival outcomes in patients with breast cancer. For the purposes of this chapter, we define "long-term survival" as 10-year overall survival (OS).

We start by reporting national data from the United States, and exploring various socio-demographic, clinical, and pathologic characteristics significantly associated with 10-year OS. Next, we perform a literature review of epidemiologic studies from the United States, and around the world, to survey for trends in this growing population. Finally, we explore numerous clinical considerations in addressing the needs of this specific population, with lessons learned from the coronavirus disease 2019 (COVID-19) pandemic, and implications for future clinical care.

#### **2. Prevalence of long-term survivorship in the United States**

On an annual basis, the American Cancer Society (ACS) provides national survival data on breast cancer cases diagnosed within the United States. With respect to long-term survival, the society published that current "relative survival rates" for women diagnosed with breast cancer are 85% after 10 years and 80% after 15 years [7]. These rates are age- and race-adjusted; supported by the provided definition of "relative survival" as the "percentage of patients who will survive their cancer for a given period of time after diagnosis…compared to survival among people of the same age and race who have not been diagnosed with cancer" [8]. Despite high heterogeneity within the breast cancer population, the ACS did not stratify longterm survival rates by other socio-demographic, clinical, or pathologic characteristics in this publication. In order to add to ACS findings, we explored the impact of these factors in more granular detail, using OS as reported by the National Cancer Database (NCDB).

The NCDB is a United States-based registry which collects de-identified clinical, pathologic, and outcomes data on approximately 70% of all cancer diagnoses in the country [9]. Data on patients with breast cancer is uploaded into the NCDB from over 1,400 facilities, accredited by the Commission on Cancer and the American College of Surgeons. At the time of this publication, survival surveillance for patients in this repository included data collected through the year 2016. Therefore, in order to ensure adequate time had transpired to capture 10-year OS, we selected a cohort of patients diagnosed between 2004 and 2006. Univariate analysis was conducted to evaluate for independent factors (e.g., age, race, ethnicity, income, insurance status, facility type, co-morbidity index, clinical stage, grade, histology, oncotype, and treatment type) exhibiting significant association with 10-year survival. Subsequently, variables significant at the univariate level were selected for inclusion within one multiple logistic regression model also predicting 10-year survival. A p-value of <0.001 was considered significant, due to the very large sample size that may overpower correlative testing. A total of n = 515,610 patients with breast cancer were analyzed in this model. The results are depicted in **Table 1**, and explained as follows.


#### *Long-Term Survivors of Breast Cancer: A Growing Population DOI: http://dx.doi.org/10.5772/intechopen.95798*


#### *Global Women's Health*


#### *Long-Term Survivors of Breast Cancer: A Growing Population DOI: http://dx.doi.org/10.5772/intechopen.95798*


**Table 1.** *Multiple logistic regression model for predictors of long-term overall survival in breast cancer in the United States, using data from the National Cancer Database (NCDB).*

#### *Global Women's Health*

#### **2.1 Overall survival by socio-demographic characteristics**

Age at diagnosis was significantly associated with likelihood of long-term OS. The age distribution of our cohort was: n = 125,657 (24.4%) <50 years old, n = 256,003 (49.7%) between 50–70 years old, and n = 133,950 (26.0%) older than 70. Long-term OS rates were similar in patients diagnosed before 50 (54.1%) compared to those diagnosed between 50 and 70 years of age (53.0%). This may highlight the relative indolence of breast cancer as a primary malignancy, particularly when diagnosed and treated at early stages. However, a large drop in 10-year OS was seen in those diagnosed after 70 (30.1%), a cohort more likely to experience acute events due to the cumulative effect of chronic comorbidities such as hypertension, diabetes, and dyslipidemia. This is supported by the life expectancy of individuals in the United States which, in 2016, was estimated to be 78.9 years [10]. The distribution of survival, by age, may differ in other parts of the world, particularly in low- and middle-income countries, or those without mass screening programs.

Racial disparities continue to be a significant major healthcare challenge. In the 1980s, a marked divergence in death rates between White and Black women with breast cancer was first noted [11]. The implementation of mass screening programs disproportionately benefited areas wherein residents had access to favorably-resourced and accredited healthcare institutions [12, 13]; these communities were predominantly White. Additionally, hormonal therapy (e.g. tamoxifen), newly introduced to systemic treatment regimens for treatment of ER+ tumors, was not appropriate for many Black women, who are more likely to present with triple negative breast cancer (TNBC) – a type of breast cancer without ER, PR, or HER2 expression, which is unresponsive to tamoxifen regimens [14]. This is elaborated upon in Section 2.2.

Race-based survival disparity peaked in 2011, with mortality rates reported to be approximately 45% higher in Black versus White patients with breast cancer [5]. Despite improvements over the last decade, race continues to be an important predictor of 10-year OS (p < 0.001), as depicted in **Figure 1**. In our analysis, using data extrapolated from the NCDB, patients of Asian descent exhibited the highest long-term overall survival rate (51.9%), followed by White (48.0%) and then Black (40.7%) patients. Beyond access to healthcare, these race-based disparities are thought to be due to the complex interplay between multiple lifestyle factors (such as alcohol consumption and smoking), extent of comorbidity (including obesity, which is associated with worse outcomes in breast cancer due to increased estrogens and inflammatory mediators [15]), and genetics. Interestingly, from our analysis, ethnicity (defined in the NCDB as Hispanic vs. Non-Hispanic) was not determined to be a significant predictor of 10-year OS, even when adjusting for

#### **Figure 1.**

*Pictorial of key predictors of 10-year OS in the United States, through analysis of the National Cancer Database (NCDB).*

relevant confounders such as age, race, comorbidity (Charlson/Deyo index), and AJCC clinical stage at diagnosis.

Measures of socioeconomic status – including annual income, insurance status, and treatment facility type – were also significantly associated with 10-year OS in this cohort (p < 0.001). Patients who were uninsured exhibited the lowest 10-year OS rates (36.8%), in contrast to patients who had private insurance (56.5%), as depicted in **Figure 1**. A study by Ko, et al., indicates that roughly half of all racial/ethnic disparity, associated with the risk of locally-advanced disease, can be attributed to insurance status as "uninsured" or "underinsured" [16]. Patients without healthcare coverage are less likely to effectively manage chronic comorbidities, including hypertension [17] and diabetes [18], which is likely a contributing factor of higher mortality observed in this subgroup. While setting (urban vs. rural) was not a significant predictor of long-term survival, facility type was (p < 0.001), with patients treated at academic cancer programs exhibiting the highest 10-year OS rate (49.0%), followed by those treated at comprehensive community cancer programs (47.7%) and those treated at community cancer programs (43.8%). This may be due to differences in time-to-diagnosis and time-to-treatment, determined by institution size, and care practices that may differ based on the accreditation of different cancer programs, as available to different communities [19].

#### **2.2 Overall survival by clinical characteristics**

As expected, the most important predictor of survival in breast cancer in our analysis was 'stage' at diagnosis, as depicted in **Figure 1**. Breast cancer stage represents the extent of spread of cancer in the body, expressed on a spectrum ranging from 0 (the earliest form, wherein cancer cells are restricted to the milk ducts of the breast, but have not invaded other breast tissue) to IV (the latest form, where the cancer has spread to another organ in the body, referred to as "metastatic"). Staging may be *clinical* (based on physical exam and imaging such as mammogram, ultrasound, or magnetic resonance imaging) or *pathologic* (based on evaluation of breast tissue and lymph nodes removed during surgery). We found that the widest disparity in long-term OS was associated with clinical stage of diagnosis (54.5% with stage 0, versus just 6.1% with stage IV disease, p < 0.001). As the majority of patients in the United States are diagnosed at early stages of disease (i.e. 0-II), this supports the positive, clinical impact of public health campaigns that target awareness of prevalence, risk factors, signs and symptoms of breast cancer.

Diagnosis of breast cancer is typically confirmed with a biopsy, during which a tumor tissue sample is sent for evaluation by specialists in pathology. Through microscopy, and the use of staining techniques, numerous pathophysiological characteristics of the neoplasm can be determined. Important among them, is the 'breast cancer subtype', referring to the molecular profile of the tumor, based on the expression of three receptors on the surface of breast cancer cells: 1) the estrogen receptor (ER), 2) progesterone receptor (PR), and the 3) human epidermal factor growth factor 2 (HER2) receptor. The combination of these receptors forms the basis for clinical decision-making regarding targeted therapy in breast cancer. For instance, if the primary tumor is ER+/PR+, 'endocrine' or 'hormonal' therapy can be administered (e.g. selective estrogen receptor modulators, or SERMs, like tamoxifen, that directly modulate these hormonal receptors, or aromatase inhibitors, which decrease the natural conversion of androgens to estrogens in the body); if the tumor exhibits HER2+ status, the monoclonal antibody trastuzumab is given to block this receptor subtype.

The NCDB did not routinely document HER2 receptor status in cases diagnosed before 2009, thus the impact of this receptor expression on 10-year OS could not be evaluated in this multivariate model. However, the analysis did include ER status and PR status, demonstrating that positivity of either receptor significantly predicted long-term survival. This finding underscores progress made in the improvement of patient outcomes as treatment modalities become more targeted (prior to endocrine therapy, non-targeted chemotherapy was the gold standard for treatment of even hormone receptor positive breast cancer). This is also strongly reflected in one of our previous analyses, indicating that patients with tumors which were negative for all three receptors (TNBC), exhibited the lowest rate of 5-year OS (71%), followed by the ER-, PR-, HER2+ subtype (77%), the ER/PR+, HER2+ subtype (83%), and a highest 5-year OS rate seen in the ER/PR+, HER2- subtype (84%) [20].

Interestingly, HER2 overexpression, occurring in around 20% of breast cancers, is associated with worse natural prognosis due to increased growth and marked metastatic potential of these tumors [21]. However, we have shown that survival outcomes in ER/PR-, HER2+ breast cancer, in the United States, have surpassed TNBC due to the advent of HER2-targeted regimens. Therefore, HER2+ status may be predictive of treatment efficacy in breast cancer. This may not be the case globally, particularly in low- and middle-income countries which may exhibit limitation in drug funding. In 2012, the Union for International Cancer Control and the Dana-Farber Cancer Institute filed an application with the World Health Organization to add trastuzumab (a HER2-targeted therapy) to the essential medications list [22], an advisory list of the minimum medicine needs for basic healthcare systems. This was not approved until May 2015 [23]. Current literature still reports trans-national disparities in the availability of HER2-targeted therapeutics, and advocates for the distribution of more affordable trastuzumab biosimilars in order to address this ongoing need [24, 25].

Pathologists will also assign a 'grade' to the tumor under evaluation using a method of classification known as the Nottingham modification of the Scarff-Bloom-Richardson system [26]. Grading in breast cancer designates how "abnormal" neoplastic cells appear, and is based on the extent of glandular/tubular differentiation, nuclear pleomorphism, and mitotic count [27]. Grade 1 tumors are "well differentiated", meaning their growth is slower and appears most similarly to normal breast tissue. Grade 3 tumors, on the other hand, are "poorly differentiated", appearing "dysplastic" (very different from normal cells) and have a higher growth potential. Grade 2, tumors have "moderate" differentiation, and fall between Grade 1 and Grade 3 in prognostic implication. While not predictive of the same breadth of overall survivorship as tumor staging, we found in our NCDB analysis that tumor grade was still a statistically significant predictor of 10-year OS: patients with Grade 1 tumors exhibited a 10-year OS rate of 51.6% (unadjusted for stage at diagnosis) versus those with Grade 2 tumors (48.5%) and those with Grade 3 tumors (44.9%). Finally, we also showed that while 'histological subtype' (referring to the tissue type a neoplasm originated from) was not a statisticallysignificant predictor of long-term overall survival (p > 0.001), the highest 10-year OS rates were seen in the most common subtypes: ductal carcinoma (47.7%, not adjusted for stage at diagnosis) and lobular carcinoma (47.3%). Patients with some rare histologies exhibited lower rates of 10-year OS, including epithelialmyoepithelial (42.1%), fibroepithelial (34.9%), papillary (30.8%), and mesenchymal (21.4%) breast cancers. The scarcity of these subtypes has limited the ability to study these unique histologies in a high-throughput manner. However, recent studies suggest that tumor histology should be considered when determining the optimal treatment approach for each patient [28–30].

#### **3. Prevalence of long-term survivorship globally**

Survival rates for breast cancer vary considerably in different parts of the world. The 5-year OS rate – which is more commonly reported and can thus be compared when controlling for confounders such as race, stage at diagnosis, age at diagnosis, etc. – varies from over 80% in developed countries, to less than 60% in low- and middle-income countries [31]. However, less is known about 10-year OS in low- and middle-income countries. We conducted a systematic search using MEDLINE, via PubMed and Google Scholar, from inception until December 2020. We included observational cohort studies also reporting OS rates if published in the English language. The search strategy involved a combination of free text searches, as well as medical subject headings (MeSH), as follows: ("Breast Neoplasms" [MeSH], OR "breast cancer" OR "breast tumor") AND ("Survival" [MeSH] OR "Survival Rate" [MeSH] OR "Life Tables" [MeSH] OR "Kaplan–Meier Estimate" [MeSH] OR "Hazard Ratio" OR "Cox regression") AND ("Cohort Studies" [MeSH] OR "Retrospective Studies" [MeSH] or "Prospective Studies" [MeSH] OR "follow-up" or "longitude").

We found n = 37 studies reporting 10-year OS rates, as presented in **Table 2**. The majority were from high income countries (n = 27, 73%), while n = 10 (27%) reported data from low- and middle-income countries. It was found that high income countries have been reporting long-term OS data over a longer period of time (1978–2020), while data from low- and middle-income countries have been published more recently (2008–2020). Additionally, cohorts used in studies from high income countries were larger (mean sample size: n = 1,573) than those from low- and middle-income countries (mean sample size: n = 268). In comparing data published since the year 2000, the mean 10-year OS rate from high-income country studies was 72%, versus the mean 10-year OS rate from low- and middleincome countries studies, which was 64%. However, these comparisons do not control for the impact of patients age at diagnosis (most studies did not report a



#### *Long-Term Survivors of Breast Cancer: A Growing Population DOI: http://dx.doi.org/10.5772/intechopen.95798*

#### **Table 2.**

*Review of global cohort data reporting long-term overall survival rates of breast cancer.*

mean age), disease stage at diagnosis (though all cohorts reported individuals from all four stages of breast cancer), race, or the presence of comorbidities in these cohorts. Therefore, more information will be needed to calculate pooled estimates of global survival, by region or country. This review of studies reveals a stark disparity in the availability of long-term outcomes data from different regions around the world.

#### **4. Forecasting stage of diagnosis in the United States to 2030**

As mentioned, the strongest predictor of 10-year survival outcomes, in breast cancer, is stage at diagnosis. As diagnostic capabilities continue to facilitate earlier identification of disease, it is important to understand how stage migration is

predicted to change in the future – an important metric for allocation of resources and services needed for this growing group of survivors.

In order to understand future stage migration patterns, in a cohort of long-term survivors of breast cancer, there is utility in forecasting the predicted proportion of cases that are expected to be early stage (0, I or II) versus late stage (III or IV) based on historical trends. To do this, we extracted annual incidence data from the NCDB from 2004–2016, stratifying by cases that were diagnosed at early stage versus those at late stages. This data was analyzed via time-series forecasting, specifically autoregressive integrated moving averages (ARIMA) modeling, which considers annual variation and accounts for temporal correlation in analysis of historical data [32]. The performance of ARIMA models has been found to be comparable to other time series models in its capacity to forecast healthcare data, such as the Bayesian shared two-component model [33].

Multiple ARIMA models were generated using the Statistical Package for the Social Sciences (SPSS) Version 27.0 software (IBM Corp, Armonk, NY) using different combinations of the autoregressive parameters for 'p', the order of the autoregressive model, 'd', the degree of differencing and 'q', the order of the moving average (p, d, q). The most predictive model was selected using the lowest Bayesian Information Criteria, and mean absolute percentage error, and this was the (0, 1, 0) ARIMA model. **Figure 2** depicts 1) the historical incidence of breast cancer in the United States (black curve), stratified by stage at diagnosis (blue for early-stage and red for late-stage) for the years 2004–2016, and 2) forecasted incidence of total, early stage and late stage cases to the year 2030. The annual proportion of new cases diagnosed at late-stage is highlighted on as an emboldened numerical figure in red. Tabulated numerical data of these forecasts can be found in **Table 3**.

We found that, based on historical trends, the proportion of cases diagnosed at advanced stages of disease is projected to fall to 10.7%, compared to the historical proportion, in 2004, of 19.8%. Based on this projected stage migration, we can expect the number of long-term survivors in the United States to continue to grow. In Section 5, we discuss the impact of the COVID-19 pandemic on mass screening, and implications for staging and care of patients diagnosed during 2020.

#### **Figure 2.**

*ARIMA forecasts of breast cancer incidence in the United States to the year 2030, stratified by stage at diagnosis, using the NCDB.*


#### *Long-Term Survivors of Breast Cancer: A Growing Population DOI: http://dx.doi.org/10.5772/intechopen.95798*



#### *Global Women's Health*

### **5. Impact of the COVID-19 pandemic**

#### **5.1 COVID-19: Epidemiology & healthcare impacts**

The COVID-19 pandemic is now a defining feature of the year 2020. This novel coronavirus was identified in 2019, as the etiology of a pneumonia diagnosis in Wuhan, in the Hubei province in China [34]. Genomic sequencing and phylogenetic analysis indicated that the coronavirus that causes COVID-19 is of the same subgenus as the severe acute respiratory syndrome (SARS) virus [35, 36]. This led to the determination that COVID-19 is due to severe acute respiratory syndrome coronarivurs-2. Following its discovery, the outbreak of this disease spread rapidly: on January 10, 2020, the genomic sequence of SARS-CoV-2 was released and shared globally by China [37]; by February of 2020, COVID-19 had quickly spread through the Hubei province [38]; and On March 11,2020, the World Health Organization, had declared the COVID-19 outbreak a global emergency and pandemic [38].

In an attempt to flatten the epidemiologic growth curve of new COVID-19 diagnoses, public health departments implemented targeted social measures to decrease transmission rates. This included emphasis on social distancing, stay-athome mandates, a requirement of face masks worn in public, and hand hygiene [39]. Additionally, in order to reduce mortality and relieve the case-load pressure on clinical care providers, many healthcare systems were forced to change clinical practice. While there has been much investigation into the pathology and biologic effects of COVID-19, the overall impact of COVID-19 on management of chronic health outcomes – including breast cancer management and overall survival – is still evolving.

Due to the COVID-19 pandemic, the mechanism for healthcare delivery has changed substantially. One of the changes seen in the United States, was the broad adoption of telemedicine and the upheaval of the in-person visit. Prior to the year 2020, the use of telemedicine was unsubstantial [40]. However, telemedicine visits increased from 1.1% during the second quarter (Q2) of 2019, to 35.3% in Q2 of 2020 [41]. Correspondingly, as the rise in the rate of remote visits increased, the number of in-person visits decreased – the number of office-based health care visits in Q2 of 2020, decreased by 50.2% compared with the previous year [41]. While helping to slow the dissemination of COVID-19, this decrease of in-person visits has made the full-spectrum of care for patients with breast cancer challenging, because physical exams and in-person evaluations have also declined. As a result, co-morbidity management may have also suffered: during Q2 of 2020, blood pressure assessments decreased by 50.1%, while cholesterol assessments decreased by 35.3% [41].

The overall effect of COVID-19 on delays in cancer diagnosis, disruptions in treatment, and modifications to therapeutic regimens is still being evaluated. One report, including 609 patients with breast cancer, identified treatment delays for 44% of the study population, aged 45 years and younger [42]. Another study suggests a higher death rate in cancer patients in receipt of recent therapy, however the proportion of patients reported on active therapy, in this study, was marginal and thus conclusive correlation cannot be determined [43–45]. Literature has shown that patients with cancer, when compared to those without cancer, are at increased susceptibility to infection, secondary to systemic immunosuppression from their cancer or anticancer therapy [46–49]. Initial reports suggested patients with cancer experienced more frequent COVID-19 complications [43, 50, 51]. As a result, physicians and patients must strategically balance the risks of cancer advancement, cancer relapse, etc. with the risks of hospitalization or death secondary to a COVID-19-related complication. Through diagnoses to management, special concern for patients with cancer is warranted due to the pandemic.

Patients with breast cancer might be at an increased risk for treatment-related complications and other health issues during the COVID-19 pandemic. The CDC reports that having cancer increases your risk of severe illness from COVID-19 [52]. Several studies have been conducted with respect to the effects of COVID-19 on patients with cancer. One multicenter study was conducted to evaluate the clinical characteristics of COVID-19-infected patients who died within 28 days of hospitalization in the intensive care unit [53]. This study reported 784 deaths after 28 days, 60 of these deaths (7.7%) were among those with active cancer; their multivariable model revealed that active cancer was associated with increased COVID-19-driven mortality (odds ratio (OR), 2.15; 95% CI, 1.35–3.43) [53]. An additional multi-institutional study was performed to evaluate the impact of COVID-19 on patients with active or prior malignancies [54]. The primary end point of this analysis was allcause mortality, within 30 days of a COVID-19 diagnosis. Within this population, 22% had hematologic malignancies, and the remainder were previously diagnosed with solid tumors [54]. This study did *not* find an association between increased COVID-19-related mortality and cancer type, anticancer therapy, or recent surgery. There were several factors associated with increased 30-day mortality: male sex (OR, 1.63 [95% CI, 1.07–2.48]), older age (per 10 years) (partially adjusted OR, 1.84 [95% CI, 1.53–2.21]), increased comorbidities (≥2) (OR, 4.50 [95% CI, 1.33–15.28]), a previous smoking status (OR, 1.60 [95% CI, 1.03–2.47]), Eastern Cooperative Oncology Group performance status 2 (OR, 3.89 [95% CI, 2.11–7.18]) or more (OR, 5.66 [95% CI, 2.79–11.47]), and progressive cancer (defined as no longer responding to treatment) (OR, 5.20 [95% CI, 2.77–9.77]) [54].

#### **5.2 Relationship between COVID-19 and breast cancer**

The increased risk of severe illness, secondary to COVID-19, in patients with breast cancer [52] might be multifaceted. Both cancer, and cancer treatment, can cause a significant physiologic strain on protective mechanisms of the human body. The immune system is intrinsically linked to breast cancer pathogenesis via inflammatory pathways, immune surveillance, and adaptive immunity [55]. Chronic inflammatory activity has been discovered in all breast cancers, regardless of breast cancer subtype [56]. This chronic inflammation can lead to damaged breast cells, which may support continued tumor progression, with some breast cancer models revealing CD4+ T lymphocytes indirectly promoting invasion and metastasis [57].

An additional reason for a potentially increased risk of serious complications, including death, secondary to COVID-19, in breast cancer patients, is impaired immunity due to chemotherapy. Treatment, with chemotherapy or radiation therapy, can lead to chronic pain, immune suppression, treatment-related toxicities, failure to thrive, and decreased physical and cognitive abilities [58]. Chemotherapy has been shown to induce neutropenia and lymphopenia in patients [59]. Women with breast cancer, who were treated with adjuvant therapy that consisted of chlorambucil, methotrexate, and 5-fluorouracil had decreased peripheral blood lymphocytes [60]. Similarly, other studies report decreased CD4+ cell counts along with concurrent pneumocystis pneumonia in patients with breast cancer who had received multi-agent chemotherapy and radiation therapy [61].

The relationship between the uses of immune check point inhibitors (ICIs) in breast cancer patients is another new area of interest during the COVID-19 pandemic. Several ICIs have been developed targeting breast cancer; some of the most clinically-advanced are those that target programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1) [62]. Anti-PD-1/PD-L1 agents are an emerging treatment modality with encouraging results for aggressive breast tumors, like

#### *Long-Term Survivors of Breast Cancer: A Growing Population DOI: http://dx.doi.org/10.5772/intechopen.95798*

triple negative breast cancers [63]. These ICI therapies, however, are associated with several significant side effects -- one of which includes inflammatory syndromes like pneumonitis, which targets the lungs [64, 65]. These treatments have also been associated with increased inflammation and tissue damage [66, 67]. Currently, reports evaluating the relationship between anti-PD-1/PD-L1 agents and COVID-19 are ongoing. But, some preclinical studies reveal that viral clearance is accelerated by PD-1/PDL-1 pathways and, thus blocked by immune check point inhibitors [68]. Other reports have associated COVD-19 with increased T-cell exhaustion when there is increased expression of PD-1 and PDL-1 [69].

As the effect of COVID-19 on breast cancer patients in receipt of ICIs is still being evaluated, members of the medical community defer to historical trends between ICIs and other viruses for clinical decision making. For instance, the checkpoint inhibitor, pembrolizumab has demonstrated efficacy in a subset of patients with progressive multifocal leukoencephalopathy caused by JC virus infection [70]. Additionally, some studies have noted that ICIs exacerbate viral lung infections, with increased toxicities observed in the winter months when the majority of the population are diagnosed with colds and the flu [71, 72].

Although there are increased risks and side effects associated with the use of chemotherapy and ICIs during the COVID-19 pandemic, the benefits of these treatment modalities could outweigh the risks. The OS of breast cancer patients has improved significantly over the last three decades, due in part to improvements in systemic chemotherapy, endocrine therapy, targeted therapy, and recently the application of ICIs [73, 74]. Therefore, while breast cancer therapies may be associated with negative side effects, recovery is possible with appropriate management, dependent upon tumor burden and the overall health status of the patient [75–77]. In order to maximize the clinical efficacy of these treatment modalities, while limiting COVID-19-related health risks, additional research is needed to guide practice.

#### **6. Clinical considerations of a growing cohort of long-term survivors**

While outcomes following treatment of invasive breast cancer have become increasingly favorable, survivors remain at-risk for recurrence of disease, either loco-regionally or at a distant site. In one large cohort of 9,514 women diagnosed with breast cancer under the age of 75, 10.4% developed distant metastasis, most commonly at a bony site [78]. Patients were more likely to experience recurrence in the period 5–10 years after diagnosis, if they presented with primary tumors that were ER-positive, lymph-node positive, or larger than 20 mm in size [78]. Women with ER-negative tumors, however, have a lower risk during this period. The development of multigene sequencing panels predicting outcomes in ER-positive tumors can guide clinicians to ensure at-risk patients receive the appropriate adjuvant therapy.

Survivors of breast cancer should undergo regular follow-up for surveillance and management of treatment-related effects, as well as breast-specific and other indicated imaging to evaluate for malignant recurrence, or new disease. This management necessarily includes a wide range of disciplines in medicine. Breast surgery or radiation therapy can result in chronic pain, fibrosis, fat necrosis, or recurrent skin infections in the chest wall [79, 80]. Patients are also at long-term-risk for cardiovascular dysfunction, including congestive heart failure [81], ovarian failure [82], and even the development of secondary cancers [83]. For this reason, the care of long-term survivors of breast cancer should be based on collaboration between multiple subspecialties. Patients should also continue to receive age-appropriate screening as indicated for the general population with respect to conditions other than breast cancer.

Cancer diagnoses are also associated with increased patient distress and anxiety [84]. Therefore, clinicians are strongly encouraged to consider psychosocial support for long-term breast cancer survivors, as an important complement to clinical monitoring. Providing integrated care that is directed to the overall wellness of the patient, maximizes the potential to increase patient satisfaction, increase patient medical compliance, and preserve quality of life [85–87]. Interestingly, it has also been found that ethnic minority groups, who typically report poorer quality of life and worse distress after diagnosis, may derive more acute benefit from integrated modalities like art therapy [88]. It is also of importance to note that effective psychosocial support programs have been shown to be significantly associated with favorable clinical outcomes [84, 89–92].

#### **7. Conclusions**

The advancement of screening modalities and novel therapies has led to more favorable prognoses in patients with breast cancer. As a result, long-term breast cancer survivors are a large and continually-growing group, globally. This group is also projected to increase, substantially, within coming years. While these trends are favorable and clinically promising, patients with breast cancer should undergo regular follow-up for surveillance and management of treatment-related effects, as well as potential disease recurrence. In the time of the COVID-19 pandemic, it is also important to note a potential combinatorial effect of possible complications secondary to cancer treatment received, and possible impact on screening and treatment delays imposed by the novel coronavirus, on both communities and health care delivery systems.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Abbreviations**


*Long-Term Survivors of Breast Cancer: A Growing Population DOI: http://dx.doi.org/10.5772/intechopen.95798*

#### **Author details**

Nadeem Bilani, Elizabeth Blessing Elimimian, Leah Elson, Hong Liang and Zeina Nahleh\* Cleveland Clinic Florida, Weston, Florida, USA

\*Address all correspondence to: nahlehz@ccf.org

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 9**

## Implication of Connexin 43 as a Tumor Suppressor in Pathogenesis of Breast Cancer

*Rabiya Rashid, Shazia Ali and Mahboob-Ul-Hussain*

#### **Abstract**

Breast cancer (BC) is a global public health burden, constituting the highest cancer incidence in women worldwide. Connexins 43 proteins propagate intercellular communication, gap junction intercellular communication (GJIC), remarkably expressed in several tumor types including liver, prostate, and breast. This domain of Cx43 possesses functionally critical sites identical to those involved in gating of channel and phosphorylation sites for various kinases. However, the mechanism by which Cx43 down regulation occurs in breast cancer is far from clear. Several mechanisms like Cx43 promoter hyper-methylation or a cancer-specific reduction of Cx43 expression/trafficking by the modulation of various components of the Cx43 life cycle give the idea to be involved in the down regulation of Connexins in mammary glands, but irreversible mutational alterations have not yet been proved to be among them. Summarily, the efficacy or specificity of these drugs can be increased by a combinatory approach considering an effect on both the Connexins and their regulatory molecules. This chapter will summarize the knowledge about the connexins and gap junction activities in breast cancer highlighting the differential expression and functional dynamics of connexins in the pathogenesis of the disease.

**Keywords:** Breast Cancer, Connexin, Tumor Suppressor, Gap junction, mammary gland

#### **1. Introduction**

Cancers that originate from the breast tissue are called as Breast cancers. Quite often, these cancers originate from the epithelial cells lining the milk ducts or lobules supplying the ducts with milk [1]. Sub-classification of breast cancer into various types is done on the basis of certain characteristics that the cancers develop depending on their origin i.e. whether cancer originates from glandular portion or ductal portion of the breast. Accordingly, cancers that stem from lobules are called as lobular carcinomas, whereas those stemming from ducts are called as ductal carcinomas. Once Primary tumors become invasive, it spreads beyond its place of origin (Breast) to the regional lymph nodes. It may also metastasize i.e., expand to different organ systems of the body, thereby becoming systemic in nature. On the basis of this expansion, breast cancer is of two types Non-invasive or in-situ and invasive. A non-invasive or in-situ cancer is one where the cancer cells

remain confined to boundaries of the lobular unit or draining duct of their origin. On the other hand, cancer cells that traverse outside the basement membrane of the lobules or ducts into the surrounding normal tissues are classified as invasive cancers. Apart from these, there are other types of breast cancer with different stages, varied aggressiveness and different genetic makeup. These factors greatly affect the chances of survival of a patient. Several breast cancers are up regulated by estrogens. These cancer cells carry estrogen receptors on their surfaces and are called Estrogen Receptor-positive cancers or ER-positive cancers. Similarly, some women suffer from another type of breast cancer called as HER2-positive breast cancer. HER2 is a gene responsible for cell growth, division and repair. Increased copy number of HER2 gene may result in faster growth of cancers. Women with HER2-positive breast cancer have higher incidence of disease recurrence making it as a risk factor for breast cancer recurrence. The disease is also more aggressive than women who do not have this type of breast cancer.

#### **2. Stages of breast cancer**

Expansion of a cancer determines its stage. Stages of a cancer indicate whether the cancer is limited to the area of origin or has spread to other healthy tissues of the body. Four important characteristics determine a cancer stage:

a.Size of the cancer.

b.Type of cancer i.e. invasive or non-invasive.

c.Has cancer reached lymph nodes,

d.Has cancer metastasized to other body parts.

Firstly, on the basis of extent of the cancer, it can be classified as local, regional or distant. A cancer is **local,** when it is confined within the breast (where it originated). It is **regional** when lymph nodes are involved. And it is **distant** when it has metastasized to other body parts as well.

There is another staging system used to describe the cancer called as TNM staging system. The TNM System is based on three components - size of the tumor (denoted by T), involvement of the lymph node (denoted by N) and whether the cancer has metastasized (denoted by M).

**Stage 0**: It is a non-invasive stage, during this stage cancer is present at its origin e.g., Ductal Carcinoma In Situ (DCIS). There is no indication of the cancer cells or non-cancerous abnormal cells traveling beyond their origin to neighboring normal tissues.

**Stage I**: This stage describes an invasive breast cancer i.e., cancer cells invade neighboring normal tissues. There are chances of microscopic invasion in this stage. In such an invasion, the cancer cells have just begun to travel outside the boundaries of their duct or lobule. However, the invading cancer cells are not more than 1 mm.

**Stage II**: This stage is further sub-categorized into stages IIA and IIB, both describing a different invasive breast cancer. Stage IIA refers to an invasive breast cancer where the tumor cannot be located in the breast but lymph nodes (axillary) under the arm show presence of cancer cells.

**Stage IIB:** Refers to an invasive breast cancer where a tumor sized between 2 cm and 5 cm has spread to axillary lymph nodes.

*Implication of Connexin 43 as a Tumor Suppressor in Pathogenesis of Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.97582*

**Stage III**: This stage is further sub-categorized into three stages - Stages IIIA, IIIB and IIIC. Stage IIIA refers to an invasive breast cancer where either the tumor cannot be located in the breast but cancer is found in axillary lymph nodes which are clumped or clinged to other structures, or lymph nodes at breast bone may be involved too.

**Stage IIIB**: Defines an invasive breast cancer stage where cancer has involved chest wall or breast skin or both and may involve axillary lymph nodes and showing Stage IIIA like features too.

**Stage IIIC**: Refers to an invasive breast cancer where there is no evidence of cancer in the breast or, in the event there exists a tumor, it is of any size, which may be involving chest wall or breast skin, or both. In this stage, the cancer has also extended to the lymph nodes below or above the collarbone and may also have spread to axillary lymph nodes or to lymph nodes near the breastbone.

**Stage IV**: This stage describes an invasive breast cancer which has extended outside the breast and adjacent lymph nodes and has affected other organs of the body e.g., lungs, bones, brain, liver, skin etc.

#### **3. Epidemiology of breast cancer**

In Modern world, occurrence of non-communicable diseases is increasing day by day [2, 3]. This is mainly due to factors like increased lifespan, prolonged exposure to risk factors and changes in lifestyle. While being one of the most crucial diseases in the world, cancer is also regarded as a complicated-on account of being multifactorial, epidemiologically. In 2012 alone, around 14.9 million new cases of cancer were tradition, culture, food habits, intra-community marriages and ethnicity. In recent past recorded. It is estimated that in the next two decades, this number will be around 22 million [4]. Now a day, breast cancer is becoming more common in women and is cosmopolitan in nature with high rate of incidence [5]. It accounts for 25% of all types of cancers, recording 1.7 million new cases per year. It is also the second common most cancer [4]. As per WHO, the latest incidence rate of breast cancer in east Africa to west Europe ranges from 19.4 to 89.7 per one lakh people respectively [6]. Other than its fast growth rate in South America and Africa, breast cancer incidence in on rise in several Asian countries too. For instance, Japan witnessed a 6% increase per year from 1999 to 2008. In Australia, mortality rate due to breast cancer has reduced by 2%. While it is increasing in several countries. Malaysia and Thailand recorded the highest increase. The ratio of mortality and incidence of breast cancer in the world and Asia-Pacific countries is 0.30 and 0.27 respectively. However, this ratio of mortality and breast cancer incidence is 0.2 and 0.41 in the Pacific and Southeast-Asian countries respectively [7]. Not so long ago, breast cancer incidence was rare in South Korea. However, now, the incidence and death rate from breast cancer has increased [8]. Hong Kong has seen a decline in the incidence [9]. Generally, different regions show different breast cancer incidence due to difference in risk factors, level of education, different life expectancy, screening programs [10], and cancer registration [2]. The number of diagnosed breast cancer cases is increasing because of the increased life expectancy and increased full health screens [10]. Cancer accounts for around 3–4 million deaths worldwide annually. Of these, 2–3 million deaths occur in developing countries [11]. In India, cervical cancer has a higher occurrence than breast cancer. On the contrary, our state (J&K) in the Indian Subcontinent, Kashmir shows a reverse trend (World Health Organization, 1978) (**Figure 1**). Rise in breast cancer in Kashmir valley is considered a major health concern. Experts attribute this increase in breast cancer to various factors like sedentary lifestyle, bottle feeding, late

#### **Figure 1.**

*Shows year-wise number of registered cancer patients, incidence sites and cancer trends 2000–2012). Adapted from Wani [12].*

marriages etc. Kashmir valley is quite different from other areas with respect to its unique geographical location, Kashmir has seen a huge increase in the occurrence of breast cancer among its unexplored ethnic population. The overall cancer incidence in Kashmir region is increasing. In men, esophagus and gastroesophageal (GE) junction, lung, stomach, colon, rectum, lymph nodes, skin, laryngopharynx, blood, prostate and brain are the common sites of cancer. While in females the common sites are breast, esophagus and GE junction, ovary, colon, rectum, stomach, lung, gallbladder, lymph nodes, blood and brain.

#### **4. Connexin 43 and breast cancer**

Connexin 43 is the most widely expressed gap-junction protein in normal breast tissue and is thought to play important role in normal mammogenesis, lactogenesis and involution [13]. Cx43 is not expressed in normal breast stem cells but is expressed in the normal breast epithelial cells derived from these breast stem cells [14–16]. Studies have shown that Cx43 is down regulated at the mRNA and protein level in human breast tumors and several human mammary tumor cell lines [17]. Decreased expression of connexin gap junctions is seen in breast cancer at various stages of progression and restoration of gap-junction intercellular communication. Studies have shown that Cx43 is down regulated at the mRNA and protein level in human breast tumors and several human mammary tumor cell lines [17]. Decreased expression of connexin gap junctions is seen in breast cancer at various stages of progression and restoration of gap-junction intercellular communication. Under in vitro conditions it has been seen that the upregulation of connexins restore normal phenotype and reduce tumor growth in vivo conditions [18, 19]. Various studies have shown that down-regulation of Cx43 plays role in primary tumor formation as well as its metastasis in breast tissue. Primary breast cancer is generally composed of tumor cells and surrounding connective tissue. The arrangement within cancer creates multiple patterns of cell–cell interactions among tumor cells and between tumor cells and normal neighboring stromal cells. Among, these patterns the GJIC involving Cx43 is considered to be initial step associated with malignant cell transformation. Studies have shown down-regulation of Connexin 43 gap-junction occurs in human breast cancer tissues compared with the non-neoplastic breast tissue surrounding primary tissue. It has been seen that re-expression of Cx43

*Implication of Connexin 43 as a Tumor Suppressor in Pathogenesis of Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.97582*

reverses the malignancy of human mammary carcinoma cells [19–21]. In, rat mammary carcinoma induced by DMBA data obtained demonstrates that connexin 43 gap junction loss is a common feature of transformed mammary neoplastic cells. Furthermore, data obtained with rat mammary carcinoma, induced by DMBA also demonstrates that the loss of connexin 43 gap junction is a common feature of mammary neoplastic transformed cells. In human mammary carcinoma (MDA-MB-435) cells it has observed that the Cx43 re-expression suppresses the cancer phenotype, increases ability of cells to differentiate into well defined 3D structures and also reduces the tumor growth in animal models [20, 22]. Studies have also shown that down-regulation of endogenous connexin 43 expression by small interfering RNA promoted a more aggressive phenotype in human breast cancer cell lines. It was seen in this study that Cx43 reduced the expression of fibroblastic factor receptor (FGFR) and of other proteins that are involved in tumor progression. Studies have revealed that over expression of Cx43 in tumor cells not only restores growth control, but they also revert to less malignant phenotypes [18]. Upregulation of Cx43 by the drugs like genistein and quercetin leads to GJICindependent inhibition of cell proliferation [23]. Cx43 plays role in tumorigenesis probably by inhibiting angiogenesis independently of cell communication as inhibition of Cx43 expression by RNAi in breast cancer Hs578t cells, resulted in faster growth and increased aggressiveness of the cells, TSP-1 expression was reduced while pro-angiogenic factor VEGF was upregulated. Similar results were observed in MDA-MB-231 cells over expressing Cx43. In addition conditioned media from these cells inhibited in vitro endothelial cell tubulogenesis and migration [24]. Additionally, tumor angiogenesis was decreased in xenografts of Cx43 overexpressing MDA-MB-231 cells. Altogether these findings suggest that Cx43 plays tumor suppressing role by mediating cell proliferation, migration and angiogenesis, enduring support to relatedness between physiological variation in Cx43 levels and aggressive of breast cancer. Tumor metastasis, a multi-step process involves the entry of transformed cells into the circulation after dissociating from the site of origin, extravasation from the vascular system and proliferation into tumor masses at secondary tissue sites. Different stages of this metastatic cascade depend both on cell–cell and cell-matrix interactions [25]. Metastasis has been shown to be promoted by down-regulation of connexins as the breast metastasis suppressor 1 gene exogenous expression in MDA-MB-435 cells led to upregulation of Cx43 and restoration of GJIC, providing evidence that connexins act as tumor suppressors in metastasis [26]. The migratory potential and ability to invade through basement membrane matrix was found to diminish in cells with exogenous expression of Cx26 and Cx43 during functional in vitro studies [27] along with a slight drop-off in matrix metalloproteinase activity [28]. Additionally, studies show decrease in the number of metastases to lungs in mice injected with breast cancer cells expressing Cx43 relative to mice injected with vector controls only [20]. The movement of cancer cells across the endothelial cell barrier as they move in and out of the blood vessels has been shown to be a key step in metastasis and studies have shown that Connexins play an important role in tumor cell vascular intravasation and extravasation [29]. Co-culturing of endothelial cells with breast cancer cells results in the reduction of GJIC in endothelial cells. This reduction weakens cell–cell contacts and also it becomes easier for cells to cross endothelial barrier during the process of extravasation and intravasation [30]. This fact was supported by another study which shows that over expression of Cx43 in HBL-100 breast cancer cells (GJIC deficient) makes them capable of forming heterocellular junctions. These junctions allow dye transfer between human microvascular endothelial cell expressing cx43 and breast cancer cells resulting in tumor cell diapedesis. Treatment of endothelial culture with GJIC inhibitors or co culturing of endothelial cells with

breast cancer cells that express mutated or non-functional Cx43 results in blockage of trans endothelial migration [31]. Hence these studies imply that both homocellular GJIC between endothelial cells and heterocellular GJIC between endothelial cells and breast cancer cells facilitate trans endothelial migration. A series of studies performed on the effects of metastatic breast cancer cells on osteoblast differentiation with MDA-MB-231 and MC3T3-E1 cells showed inhibition of osteoblast differentiation by conditioned medium from breast cancer cells [32, 33]. It has also been demonstrated by other studies that in MDA-MB-231 Cx43 expression results in decreased expression of OB-cadherin [34] a similar trend was also found in Cx43 expressing MDA-MB-435 cells. Decrease in the expression of N-cadherin, a protein which is involved in increased motility, invasion and metastases of breast cancer cells [35, 36] has been observed in in Cx43 over expressing MDA-MB-231 cells [34] this clearly shows that it contributes to decreased metastasis in vivo. In human glioblastoma cells it has been seen that Cx43 enhances response to chemotherapeutic agents or low serum hence confirming the fact that Cx43 shows anti metastatic effect [37]. In human breast cancer tissue, studies have also demonstrated that expression of Cx43 is directly correlated with the expression of BAK (Bcl-2 homologous antagonist/killer), a pro-apoptotic gene of the Bcl-2 family [38]. In human mammary carcinoma cell, MDA-MB-435 Cx43 suppressed the cancer phenotype and cell growth in culture and in animal models. There remains little doubt that down regulation of Cx43 plays a very important role in the primary tumor formation and its metastasis in mammary glands. However, the mechanism by which Cx43 down regulation occurs in breast cancer is far from clear. Several mechanisms like Cx43 promoter hyper-methylation or a cancer-specific reduction of Cx43 expression/trafficking by the modulation of various components of the Cx43 life cycle appear to be involved in the down regulation of Connexins in mammary glands, but irreversible mutational alterations have not yet been proved to be among them. Therefore, the role of Cx43 in carcinogenesis requires further investigations. Additional studies on Cx43 in different cancers, thus, will establish its role in cancer signaling and thus as a therapeutic target.

#### **5. Regulation of Connexin 43 by epigenetic mechanisms and transcription factors**

Tumors and transformed cell lines generally exhibit down regulation of Connexin expression. This down regulation is said to be responsible for the loss of proliferating control. However, deletion or mutation of connexin gene as a common factor in human tumors has not yet been demonstrated by various intensive studies on the subject. On the other hand, what various studies have shown is that silencing of Connexin expression in several kinds of malignant cells can be caused due to epigenetic inactivation of the promoter region by hypermethylation. Studies have also indicated that types of cells and connexins determine the effects of DNA methyltransferase inhibitors on connexin expression, as illustrated by Vinekn et al. in a review [39]. A correlation was established with micrometastasis into lymph nodes and the lack of Cx43 mRNA expression in adjacent normal lung cancer tissue in human non-small lung cancers [40]. Patients lacking Cx43 mRNA possessed higher frequency of promoter methylation compared with Cx43 mRNA-positive patients, as reported by Chen. Their data also indicates a possible interference of promoter methylation with AP-1 binding to the promoter which results in lack of Cx43 gene expression. The human Cx43 proximal promoters possesses several binding sides for Sp1 and AP1 transcription factors and have been demonstrated to be indispensable for optimal promoter activity by promoter/report assays and

*Implication of Connexin 43 as a Tumor Suppressor in Pathogenesis of Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.97582*

Sp1/AP1 over expression studies. The Sp1- and Ap1- binding sites were shown to contribute to the activity of the promoter. Each of them also contributed to bind the transcription factors Sp1/Sp3 or AP1, respectively. Both Sp1 and Sp3 resulted in the rat Cx43 promoter activation during trans-activation assays. These findings indicate the importance of the transcription factors Sp1, Sp3 and AP1 in rat Cx43 proximal promoter activity. Cell type-specific expression of Cx43 may thus depend on additional activators or repressors in different Cx43-expressing cell types (including cardiomyocytes) as similarities exist in proximal promoter regulation by universally expressed transcription factors (Sp1, Sp3, AP1). Although the mechanism Connexin gene silencing by DNA methylation is clear, the origin of this epigenetic modification still remains elusive. In liver cancer, elevated DNMT1 mRNA levels are thought to decrease expression of connexins, in casu Cx26 [41]. Moreover, the aberrant binding of transcription factors to methylated Connexin gene promoters may contribute to poor Connexin expression in cancer cells. This is supported by the decreased Cx43 gene transcription accompanied by DNA methylation in human non-small cell lung cancer cells. The decreased Cx43 gene transcription is also correlated with reduced binding of activator protein 1 (AP1) to its promoter [40]. Furthermore, in human breast cancer cells [42] and rat liver cancer cells [43] the Sp1 cis-acting elements of the Cx26 and the Cx32 gene promoter are especially rich in methylated CpG dinucleotides.

#### **6. Regulation of Connexin 43 by micro RNAs**

Almost one-tenth of all new cancers and 23% of cancer cases detected in females are breast carcinomas with more than 1 million diagnoses every year worldwide [44, 45]. Major causes of this disease-related death are relapse and metastasis [46, 47]. Recent studies that on the metastatic mechanisms of breast cancer suggest the gap junction to be a major regulator of tumor metastasis [48]. Located at the cell membrane, the gap junction primarily comprises of different connexin proteins. These connexin proteins are closely associated with numerous functions of the cell [49, 50]. Connexins constitute a family of 21 members with Cx43 being abundantly expressed in the mammary gland [49]. It is reported that Cx43 plays an important role in normal cell migration [51] and tumor cell invasion [52]. As such, promising strategies in regulating cell functions are provided by the regulation of Cx43 expression [53, 54]. Different transcription factors tightly regulate the expression of CX43 gene at transcription level. Studies have found that Sp1 (specificity protein 1), Sp3, AP-1 (activating protein 1) and c-Jun can promote transcriptional activity of Cx43 gene by directly binding to its promoter [55, 54] addition, at the posttranscription level Cx43 is also closely regulated by miRNAs [53, 56, 57]. miRNAs, largest groups of posttranscriptional regulators, [58]. Eight bases at the 5′end of miRNAs, are involved in posttranscriptional regulation. These two to eight bases could bind to the 3′-UTR of the target genes in order to bring about inhibition of gene expression at mRNA level [58]. By virtue of their direct or indirect regulation of target gene expression, miRNAs regulate a number of biological processes. The processes include cell cycle [59], growth [60], apoptosis [60], differentiation [61] and stress reaction [62]. Studies have identified miR-1, miR-206, and miR-381 as potent suppressors of Cx43 [53, 56, 63]. Cx43 has been found to enhance metastasis in breast cancer cells. It has been proven to be a direct negative target for miR-206, miR-1 and miR-133 and an indirect target for miR 381 [8]. During the myoblast differentiation in vitro and in vivo, two related miRNAs, miR-206 and miR-1, cause inhibition of Cx43 protein expression without altering Cx43 mRNA levels [63]. Further it has been reported by Anderson et al. that Cx43 mRNA contains

two binding sites in its 3'UTR for miR-206/miR-1, both of which are essential for an efficient down regulation. Also, they observed sections of eight nucleotides in the 3'UTR of Cx43 gene that are complementary to the first eight nucleotides from the 5′ end of miR-1. Which then they proved that miR-1 binds to these nucleotide sequences. miR-1 was also shown to cause reduction of Cx43 levels in isolated neonatal rat ventricular myocytes in culture [64]. They further found two putative target sequences in the 3′ UTR of Cx43 for miR-206 and proved that miR-206 that is expressed ectopically binds these sites. Moreover, the ectopic expression of miR-206 downregulated the endogenous expression Cx43 gene without affecting Cx43 mRNA expression. The continuous expression of miR-206 in osteoblasts resulted in decreased expression of osteoblast differentiation and Cx43 protein expression. The suppression of Cx43 gene expression was caused by miR-381 via the promoter region −500/−250 miR-381 could directly bind the sequences CACUUGUAU in the 3′UTR. Site-directed gene mutation was done (CCAAT/enhancer-binding protein α) in order to inhibit C/EBPα expression. By binding it to a canonic element (AATTGTC) located at −459/−453 in the promoter region of the Cx43 gene, they identified C/EBPα as a novel transcription factor. Therefore, miR-381 causes C/ EBPα dependent Cx43 suppression in breast cancer cells.

#### **7. IRES mediated regulation of Connexin 43**

Connexin 43 (Cx43) is one of the main gap junction (cell–cell channel) proteins expressed in the heart ventricle. Constitutive expression of Connexin 43 has been found to be responsible for the anisotropic propagation of action potentials in the heart [65]. And also, Cx43 gap junctions are essential for the synchronous contraction of the myometrium of the uterus during labour pain. While the expression of Cx43 is ordinarily sparse in the myometrium, the ovarian hormones and mechanical stretch upregulate it [66]. This upregulation is seen at the transcriptional as well as the translational level, as there is accumulation of Cx43 mRNA before the swift advent of Cx43 protein, just prior to childbirth [67–70]. The Cx43 gene like most of the connexin genes consists of two exons separated by a large intron. Exon 1 contains most of the 5P-untranslated region (5P-Utr) while the remaining 13 bases of the 5P-UTR followed by the entire coding region and the 3P-UTR are contained in Exon 2. There is wide acceptance of observation that in eukaryotes, protein synthesis initiation begins with the binding of the small ribosomal subunit to the 5P-cap structure. Then the mRNA is scanned by the 40S ribosome until it encounters an AUG codon where the 60S ribosomal subunit joins, and hence the translation begins. Between the cap structure and the first AUG codon, most cellular mRNAs contain fewer than 50 nt between the cap structure and the first AUG codon but the 5P-UTR of Cx43 mRNA has been found to 208 nt. In addition, the 5P-UTR of Cx43 mRNA has a stable secondary structure. The scanning of the 40S ribosome can be inhibited by such structures. The secondary structures of the Cx43 IRES and most of the other described IRES elements have a semi-conserved Y-like structure, which is suggested to have role in the IRES mediated translation in eukaryotic cells in Stress conditions. Inhibition of cap-dependent translation is one of the cellular responses to stress [71]. This inhibition allows continuation of synthesis of proteins essential for survival and stops the synthesis of non-essential proteins. Illustrations for this are as follows: VEGF is translated in response to hypoxia [72], the translation of the chaperone proteins Bip [73] and hsp70 takes place under conditions of cellular stress in response to misfolded and degraded proteins and in the infarcted myocardium FGF-2 functions in the salvage of cells [74]. In all these genes, IRES elements have been found that are translated even under stress conditions. The need *Implication of Connexin 43 as a Tumor Suppressor in Pathogenesis of Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.97582*

is to maintain intercellular communication via Cx43 channels even under certain stressful conditions likely. For instance, in the hypoxic hear gap junctional remodeling occurs [75] requiring the synthesis of new Cx43. Recent reports have claimed that in addition to estrogen, mechanical stretch is required to upregulate expression of Cx43 in the uterus at the commencement of labor [66]. The fetus grows faster than the uterus during the later phase of pregnancy which causes physical stretch in the myometrium. As such, Cx43 must be speedily upregulated during this time. A mechanism by which a high level of translation can be accomplished during this stress may be offered by the IRES.

#### **8. Carboxy terminal domain of Connexin 43 and human breast cancer**

Cellular communication is paramount for tissue/organ homeostasis in multicellular organisms. Exchange of small ions, secondary messengers and small metabolites required for electrical and bio-chemical coupling between cells is mediated via intercellular channels known as gap junctions [76, 77]. Each gap junction is formed by association of Connexin proteins. Human genome contains 21 different Connexin genes, expressed differentially in various types of cells and tissues [78]. Among these gap junction proteins, connexin 43 (Cx43) is major gap junction protein which is widely expressed across tissues and besides its role in mediating cell to cell communication, it also plays very critical role in cellular proliferation [79]. More precisely, Cx43 acts a tumor suppressor [80] usually downregulated in various diseases such as cancer [81, 82], connexin 43 possesses long cytosolic C-terminus and most of the non-canonical functions of connexin 43 are attributed to it [83] (**Figure 2**).

More interestingly independent of full length Cx43, CT-Cx43 expression has been found to occur in various cell types [85]. This CT domain is subjected to various post translational modifications like phosphorylation, S-nitrosylation and

#### **Figure 2.**

*Gap junctional intercellular communication (GJC) mediated by connexin proteins. Hexameric arrangements of connexin monomers comprise a hemi-channel or connexon. Adjoining cells each contribute one connexon to form a complete gap junction channel. For several connexin types, the assembly, gating and turnover of this channel are regulated to a large extent via phosphorylation of the cytoplasmic tail by various cellular kinases including: Src, PKC and MAPK. Adapted from (king and Bertram, 2005) [84].*

truncation [86]. Also CT-Cx43 has been shown to interact directly and indirectly with microtubules and actin respectively. The later takes place by the interaction of CT-Cx43 with adaptor proteins such as zonula occludens-1 (ZO-1) and drebrin (developmentally regulated brain protein). Perturbation of this interaction has been implicated for the development of various developmental and cardiac defects (**Figure 3**) [87].

The growth suppressing effect of Cx43 was not compromised while expressing only CT-Cx-43 in HeLa cells [89] and HEK-293 cells [89]. CT-Cx-43 has been shown to have nuclear localization implying that it may be involved in regulating gene expression directly or indirectly within the nucleus [89]. In direct regulation it may act as a transcriptional activator or repressor of target genes however, in indirect regulation it may regulate target gene by acting as a transcriptional activator or repressor of miRNAs targeting them. In various cancers it has been shown the expression of one tumor suppressor gene can rescue the expression of other tumor suppressor gene as well [75, 90]. However the exact mechanism is not fully understood. P53 known to act as guardian of genome is a tumor suppressor playing very important role in regulating cellular process [91] such as cell proliferation [92, 93]. The expression of p53 increases under stress conditions [94] than its basal levels under normal condition [95]. These stress conditions include DNA damage [96] and oncogenic insults [97]. Dysregulation of p53 is considered to be initiator of tumorigenesis which includes its down regulation or mutation [98–100]. Expression of p53 has been found to be upregulated by CT-Cx43 in cardiomyocytes [88]. In addition, a group of small RNAs, i.e., microRNAs (miRNAs), has been shown to be able to regulate the expression of genes implicated in various normal and pathological conditions, including cellular proliferation and cancer [101–106]. More precisely a conserved homolog of *C. elegans* miRNA lin-4 namely miR-125b has been found to be dysregulated in various cancers [107]. The expressional studies of miR-125b in various cancers have revealed that miR-125b is upregulated in some cancers and

#### **Figure 3.**

*Channel dependent and independent mechanisms by which Connexin expression can alter other genes. (A) Channel dependent mechanism. In this model, signaling molecules (red arrow) are directly exchanged between cell cytoplasms there by coordinately regulating gene expression patterns in the nucleus (N) (B) connexin dependent but channel independent mechanism. In this model connexins that may or may not be at junction membrane either bind a molecule with transcriptional activity (purple trapezoid) or can cleave such a portion of carboxy terminus to signal to the nucleus In this model connexins that may or may not be at junction membrane either bind a molecule with transcriptional activity (purple trapezoid) or can cleave such a portion of carboxy terminus to signal to the nucleus. Adapted from (Kardami et al., 2007) [88].*

downregulated in others such as breast cancer. Therefore, it has occasionally been labeled as a tumor suppressor. Most of the dysregulated miRNAs has been shown to target tumor suppressor genes such as PTEN, RB, Cx43 and p53 [108, 109].

#### **9. Connexin 43 as theurapatic target**

Connexins have a dynamic role in the metastatic process, involving multiple factors. Metastasis is preceded by a series of events -tumor cells leave the primary tumor, move too far off sites and some start secondary tumors. While dealing with therapeutic issues, this variety of roles of connexins and GJIC in tumor development requires special attention. The anti-tumor growth function of connexins and their observed loss in cancer has made it clear that a possible strategy to inhibit tumor growth could be provided by restoring connexins expression. The targets of anti-cancer therapeutics are enzymes that affect global gene expression including HDAC, a set of enzymes involved in chromatin remodeling. Upon generation and testing of many HDAC inhibitors (HDACi), it has been observed that the effects of these drugs (at least some part of them) are GJIC-dependent. When prostate cancer cells were treated with the HDACi Trichostatin A (TSA) restoration of both Cx43 expression and GJIC takes place [110]. Hyperphosphorylation and degradation of Cx43 was also countered via the modulation of MAP kinases and Src [111]. Proteins that are involved in tumor progression and metastasis can regulate Connexin expression at transcriptional level. Cx43 is transcriptionally upregulated by the Ras–Raf-MAPK pathway via the interaction of its promoter with a protein complex that contains both HSP90 and c-Myc [112]. Protein AML1-ETO fusion protein transcriptionally upregulated Cx43 expression resulting from the chromosomal translocation t(8;21) frequently associated with acute myeloid leukemia (AML) via the JNK signaling pathway. The JNK specific inhibitor SP600125 was shown to inhibit this effect [55]. Melanoma metastasis was promoted by the protease-activated receptor-1 (PAR-1) via transcriptional regulation of Cx43 [54, 113]. The importance of Connexin phosphorylation, especially Cx43, in the regulation of their levels and functions has been extensively investigated [114]. The stability and degradation of Connexin proteins are regulated by the lysosomal and proteasomal systems, in addition to phosphorylation [113]. The efficacy of drugs could be improved by the stabilization of the Cx43 protein. For example, while sensitizing cells to the pro-apoptotic effect of MG132, the rate of degradation was decreased by treatment with the proteasome inhibitor MG132 [115]. A major regulatory event in the life of Connexins is phosphorylation by the kinase Src. Phosphorylation by Src takes place either directly or via signaling intermediates such as PKC and MAPK which resuls in a disruption of GJIC [116]. This effect has been shown to lead to drug resistance [117]. In colon cancer cells that already express Cx43 mRNA, Cx43 expression and phosphorylation were enhanced by Kaempferol, a plant flavonoid, via a Stat3 dependent mechanism. However, Kaempferol showed no effect in cells that were devoid of Cx43 mRNA and deficient in GJIC [118]. Therefore, targeting the posttranslational modification of connexins is limited by the requirement of a functional transcriptional regulation. Such an isolated treatment would not be useful unless in combination with other treatments such as methylation- or acetylationmodulatory agents in order to unblock the transcription of c bvonnexins. In ovarian cancer cells, Cx43 phosphorylation and inhibition of GJIC was brought about by their treatment with endothelin-1 (ET-1), a ligand for the ETA receptor (ETAR), which is overexpressed in ovarian carcinoma [119]. The selective ETAR antagonist BQ 123, the tyrosine kinase inhibitor tyrphostin 25 or the c-Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyramidine (PP2) blocked this

#### *Global Women's Health*

effect, which suggests a role for Src in this mechanism [119]. Further, inhibition of ovarian tumor growth in vivo alongside a reduction of Cx43 phosphorylation was caused by ABT-627, an ETAR antagonist [119]. Summarily, the efficacy or specificity of these drugs can be increased by a combinatory approach considering an effect on both the Connexins and their regulatory molecules. In conclusion, gap junctional intercellular communication mediated by Connexins offer immense therapeutic opportunities that are still widely open. This approach is supported by original tools in the form of new findings regarding the regulation of Connexins expression. In view of the vast array of data about Connexins generated in various different tumor models and contexts, it is perhaps the right time for a consensus meeting devoted to focusing attention of the possibilities for Connexins as therapeutic targets.

### **Author details**

Rabiya Rashid1 \*, Shazia Ali2 \* and Mahboob-Ul-Hussain1 \*

1 Department of Biotechnology, University of Kashmir, India

2 CSIR IIIM, Srinagar, India

\*Address all correspondence to: w.shazia@gmail.com, mahboob@uok.edu.in and rashid.rabiya@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Implication of Connexin 43 as a Tumor Suppressor in Pathogenesis of Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.97582*

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### *Edited by Zhengchao Wang*

This book provides a comprehensive overview of the latest women's health research to benefit the global population of women. This book includes four sections on: "Maternal Mortality and Life Quality," "Pregnancy and Preterm Labour," "Papillary Neoplasm and Melanoma," and "Pathogenesis of Breast Cancer." It provides a comprehensive overview of the current state of the art in global women's health, focusing on the most important evidence-based developments in this critically important area.

Published in London, UK © 2021 IntechOpen © MishaBeliy / iStock

Global Women's Health

Global Women's Health

*Edited by Zhengchao Wang*