**5. Hypoxia in TNBC**

A great number of scientific studies have shown that the development of different TNBC forms is closely associated with the induction of various signaling pathways and that TNBC cells show greater sensitivity to different drugs. Recent studies showed hypoxia-inducible factor-1α (HIF-1α) was strongly correlated to clinicopathological features in many types of cancers. This molecule seems to play a significant role in the development of different tumors and breast cancer among them.

currently being tested as treatment for metastatic TNBC in combination with chemotherapy

TNBCs expressing the androgen receptor (AR-positive) account for about 10% of all TNBCs and are characterized by a more benign course [13]. These tumors express the AR, which can be detected by immunohistochemistry or the analysis of AR gene expression. AR-positive TNBCs have several common features with ER-positive breast tumors, including the expression of several estrogen-dependent genes and the frequent presence of PIK3CA mutations. Anti-androgens have been studied as potential drugs for these cancers. Few TNBCs express AR, and patients with AR-positive tumors were qualified for clinical trials with anti-androgens. Many molecules have been studied, but data on bicalutamide and enzalutamide are most extensive. Unfortunately, few patients responded to the treatment with these agents. Nonetheless, 20–35% of patients achieved disease stabilization [36]. It remains unclear whether these findings reflect the relatively mild nature of AR-positive TNBCs or whether they reflect the actual,

New treatment targets for patients with cancer are being studied. These include, among others, reparixin (inhibitor of interleukin-8 activation of CXCR1/CXCR2 chemokine receptors), CXCR1/2 (stem cell pathway), cyclin-dependent kinases, c-Met pathway, aurora kinase inhib-

After over 20 years with relatively few new treatments for the TNBC, recent years have seen a growing interest in the TNBC among researchers. This is because more and more people with breast cancer have the TNBC, which is aggressive and tends to metastasize. Currently, studies are assessing different chemotherapy regimens and are testing the usefulness of new targeted therapies. In the early stages of the TNBC, standard neoadjuvant chemotherapy might save patients' lives; patients who receive standard neoadjuvant therapy can later receive adjuvant chemotherapy or be included in clinical trials if there is extensive residual cancer after neoadjuvant therapy. Growing evidence supports the benefit of adding cisplatin to the chemotherapy with taxanes/anthracyclines in patients

Because many new targeted therapies for the TNBC are assessed in ongoing trials, we hope

A great number of scientific studies have shown that the development of different TNBC forms is closely associated with the induction of various signaling pathways and that TNBC cells show greater sensitivity to different drugs. Recent studies showed hypoxia-inducible

itor, death receptors, and CSF1 inhibitor (*colony stimulating factor 1*).

or with immune checkpoint inhibitors [35].

**4.6. Androgen-targeted therapy**

86 Breast Cancer and Surgery

but limited, activity of anti-androgens.

**4.7. Other agents**

with *BRCA* mutations [37].

**5. Hypoxia in TNBC**

that the treatment of TNBC will soon be improved.

HIF-1α is responsible mainly for cellular adaptation to hypoxic conditions; therefore, genes triggered by this factor are responsible mainly for the improvement in oxygen supply (by increasing angiogenesis, broadening the lumen of existing vessels, increased erythropoiesis or increased iron consumption), adaptation of cells to anaerobic metabolism conditions as well as for other changes facilitating cell survival in insufficient oxygen availability and modifying the main metabolic pattern. Stimulation of angiogenesis promotes the increased risk of distant metastases. Better accessibility of blood vessels increases the chance of tumor cells finding their way into the bloodstream and being transported to niches allowing settlement and formation of a metastatic lesion [38].

Hypoxia-inducible factor 1 is a master transcriptional regulator of genes regulating oxygen homeostasis. The HIF-1 protein is composed of two HIF-1α and HIF-1β/aryl hydrocarbon receptor nuclear translocator subunits. The prognostic relevance of HIF-1α protein overexpression has been shown in breast cancer. The impact of HIF-1α alternative splice variant expression on breast cancer prognosis in terms of metastasis risk is not well known.

Therefore, Dales et al. [39] investigated the prognostic value of different HIF-1α transcript expression levels in breast cancer and found a significant relationship between either clinicopathological characteristics or patient metastasis-free survival. They proved mRNA expression of a *HIF-1αTAG*splice variant reflects a stage of breast cancer progression and is associated with a worse prognosis [39].

Due to the fact that TNBC frequently shows morphologic evidence of hypoxia (central fibrosis and necrosis) [40, 41] an augmented expression of HIF-1α in tumors with a triple-negative phenotype should be anticipated. In fact, this had been elegantly demonstrated through the preferential expression of HIF-1α in peri-necrotic tumor cells in TNBC and BRCA1 mutated breast cancers [42].

In contrast, Tan et al. [43] and Choi et al. [44] demonstrated in TNBC an increase of carbonic anhydrase IX, a downstream product of the hypoxic pathway, rather than an increase in HIF-1α per se. The authors did not dispute the likely contribution of hypoxia to the tumors' aggressive phenotype.

HIF-1α overexpression is an indicator of poor prognosis and significant survival time reduction in patients suffering from breast cancer [45]. HIF-1 upregulates transcription of angiogenic genes like erythropoietin (EPO) and vascular endothelial growth factor (VEGF), which induce sprouting of new vessels and in result they increase the risk of metastasis because they boost surface of contact between tumor cells and vasculature. HIF-1 induces transcription of cytoprotective proteins in malignant cells in hypoxic conditions. HIF-1α predicts poor prognosis breast cancer [46, 47].

The relationship between inflammation and tumor progression is widely accepted. This phenomenon is also well known in breast cancer, and is mediated by numerous interleukins. Besides playing a central role in the induction of inflammatory processes, interleukin 1β (IL-1β) was also identified as a factor important for progression of the tumor and stimulation of angiogenesis as well as being responsible for the increase in the invasiveness of cancer lesions. Recently, there has been considerable interest in understanding the non-hypoxic upregulation of the hypoxia-inducible factor HIF-1α by IL-1 in neoplastic cells since aberrant expression of HIF-1α correlates with tumor progression. Naldini et al. [48] studied the effect of IL-1β on cell migration and HIF-1α accumulation in the human invasive breast cancer cell line MDA-MB-231.

Routine tests were performed in order to determine immunohistochemical expression of basic profile of diagnostic markers, such as estrogen receptor (ER), progesterone receptor (PR) and HER2. Monoclonal antibodies against receptors for estrogen (Monoclonal Mouse Anti-Human Estrogen Receptor alpha, 1:50 dilution, Clone: 1D5, Code: IR654, DAKO) and progesterone (Monoclonal Mouse Anti-Human Progesteron Receptor, 1:400 dilution, Clone: PR636, Code: IR068, DAKO) were used in order to determine the expression of steroid receptors. Evaluation of the immunohistochemical markers was performed by two pathologists as follows: ER and PR were categorized as negative—(0%), low positive—(1–10%); nuclear

Triple-Negative Breast Cancer: Expression of Hypoxia-Inducible Factor 1α in Triple-Negative…

http://dx.doi.org/10.5772/intechopen.75354

The study was conducted as follows: sections were incubated at 60°C overnight and subsequently dewaxed. The next step involved revealing the epitope by heating the slides in a buffer for 40 min. Subsequently, preparations were left at room temperature for 20 min. Preparations were rinsed in buffer and endogenous peroxidase was blocked by washing in 3% H2

10 min. In the next step, preparations were incubated with an appropriate antibody for 30 min. After incubation, preparations were rinsed in buffer for 10 min, and then incubated with the reagent (Visualization Reagent) for 30 min. After incubation with the reagent, preparations were washed in TBS (Tris-Buffered Saline, Code: S1968) with pH 7.6 for 10 min, and then incubated with 3,3′-diaminobenidine (DAB) (Substrate—Chromogen Solution) for 10 min to visualize the color of the reaction. At the end of the procedure, preparations were stained with hematoxylin.

HER2 expression was determined using HerceptTest™ DAKO test (Code: K5204). It enabled detection of HER2 expression using a polyclonal antibody against this protein (Rb A—Hu HER2—Rabbit Anti-Human HER2 Protein). Antigen retrieval for HER2 using HerceptTest was performed by immersing and incubating the slides in 10-mmol/L citrate buffer in a calibrated water bath (95–99°C) for 40 min (±1 min). After decanting the epitope-retrieving solution, sections were rinsed in the wash buffer and later, soaked in the buffer for 5–20 min before staining. The slides were loaded onto the autostainer using the HercepTest program, as described in the manufacturer's insert. In the autostainer, the slides were rinsed, placed in 200 μL of peroxidase-blocking reagent for 5 min, rinsed, placed in 200 μL of primary anti-HER2 protein (or negative control reagent) for 30 min, rinsed twice and immersed in 200 μL of substrate chromogen solution – DAB for 10 min. The slides were counterstained with hematoxylin and finally coverslipped. HER2 results were determined based on the maximum area of staining intensity according to the instruction in the package insert and the ASCO/CAP guidelines as follows: strong, circumferential membranous, staining in >30% of invasive carcinoma cell was scored 3+, moderate, circumferential, membranous staining in ≥10% of invasive tumor cells or 3+ staining in ≤30% of cells was designated as 2+ staining, weak and incomplete membranous staining in invasive tumor cells was scored 1+ and no staining was scored 0.

A total of 162 cases of breast cancer with metastasis to lymph nodes were assessed for expression of HIF-1α (Monoclonal Mouse Anti-Human HIF-1α 1:50 dilution, Clone:28b, Santa Cruz Biotechnology®, Inc.). A visualization system ImmunoCruz™ Mouse ABC Staining System (Santa Cruz Biotechnology®, Inc.) was subsequently applied; tumor-cell immunoreactivity was scored according to both the extent of nuclear staining—relative number of HIF-1α

O2 for 89

staining in >10% of tumor cells was considered positive for ER and PR.

Tumors with 0 and 1+ staining were considered negative.

It was found that hypoxia-independent induction of HIF-1α by IL-1β was associated with an increase in cell migration and a simultaneous increase in the activity of phosphorylated p38 MAPK and CXCR1 expression. Inhibition of HIF-1α by siRNA led to a significant reduction in CXCR1 expression and cell migration, confirming the role of HIF-1α in hypoxia-independent, IL-1β-induced migration of the MDA-MB-231 line cells. The results of the studies present a new role of IL-1 in breast cancer. The therapeutic approach focused on inhibition of IL-1β activity appears to be a new target for the research aimed at the development of novel methods to treat invasive breast cancer [48].
