**7. Breast cancer signaling pathways**

[154]. Recent mouse models have contributed significantly to the understanding of breast cancer, however further research into suitable animal models will be required to advance development of new therapies for breast cancer. The growth and metastasis of human breast cancer cell lines *in vivo* allows the measurement of gene function relative to disease progression and has provided much insight into the use of investigational drugs intended interrupt or

Table 3 provides an overview of the defining characteristics of established breast cancer cell lines (mouse and human) commonly used in both *in vitro* and transplantation models.

It is unlikely that any one transplantation model will ever replicate the complexity of the whole cancer process; however studies to date demonstrate that xenographs are relevant in human breast cancer. Treatment with Herceptin was shown to improve the anti-tumor activity of paclitaxel and doxorubicin against HER2/neu-overexpressing human breast cancer xenografts leading to consecutive favorable clinical trials [169, 170]. Davis *et al.* [2004] reported the effective inhibition of tumor growth and metastasis in an orthotopic xenograft model by the use of combination therapy of paclitaxel and neutralizing antibodies targeting vascular endothelial growth factor receptor 2 (VEGFR2] [171]. The results of this research likely led to the development of bevacizumab, a humanized monoclonal antibody that targets vascular endothelial growth factor A (VEGF-A) [172]. Although, bevacizumab was removed as a breast cancer indication by the FDA [173], this is yet another example of how transplantation models lead to further development in the clinic. While these are merely a few cases for breast cancer and the use of xenograft studies, the information obtained from such has been translated into successful clinical trials for a variety of cancers [174-178]. Furthermore, useful information has been gained from transplantation models with respect to toxicity and in the identification of

bone

[149]

**Cell line Species ER Status PR Status Metastasis Location Ref.** 4T1 Mouse + + Lymph node, blood, liver, lung, brain,

BT-474 Human + + Bone [156] FII3 Mouse + + Lung [157, 158] MCF-7 Human + + Lymph node, lymphatic vessel [159, 160, 161] MDA-MB-231 Human − − Lung, liver, brain and bone [162, 163] MDA-MB-435 Human − − Lung [164] MDA-MB-453 Human − − Bone [165] SUM1315 Human − − Lung, bone [166, 167] SUM149 Human − − Lung [167] T47D Human + + Lymph node, lymphatic vessel [168]

**Table 3.** Breast Cancer Cell Lines for *in vitro* and transplantation models

predictive biomarkers.

interfere with tumor growth [155].

358 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

Each of the identified breast cancer subtypes and gene expression patterns are dependent on different oncogenic pathways [105, 182]. The maintenance and differentiation of normal breast tissue is controlled by many signaling pathways and involves cytokines and chemokines, growth factors, steroid hormones, integrins, adhesion molecules and their respective receptors [183]. The regulation of such by single or combined components of the tumor microenviron‐ ment (such as fibroblasts, macrophages / lymphocytes, endothelial cells, vessels and proteins of the extracellular matrix (ECM) and stroma) have been implicated in various ways in the promotion, growth invasion and metastasis of breast cancer [184]. Cross talk or communication between the cancer cells and the factors within the tumor environment, including secretion factors from the tumor itself, can modify expression and signaling [184]. Of the several pathways indicated to play a role in cancer and CSC self-renewal, Notch, Wnt/Beta(β)-catenin, and Hedgehog (Hh) have been identified in human mammary cancer [124, 185, 186]. Addi‐ tionally, evidence has mounted to strengthen the link between nuclear factor kappa-B (NFκB), stem cells and breast cancer as elegantly reviewed in a recent paper by Shostak and Chariot (2011) [187].

#### **7.1. Notch pathway**

Four Notch proteins, namely Notch-1 to Notch-4, are expressed as transmembrane receptors in a variety of stem/progenitor cells [188, 189]. The binding of specific surface-bound ligands are responsible for triggering cleavage events at the Notch proteins by ADAM (A Disintegrin and metalloproteinase domain-containing protein) protease family and γ-secretase [188-191] causing the intracellular domain of Notch to be released and translocate to the nucleus. Once in the nucleus, downstream target genes (including c-Myc, cyclin D1,p21, NF-κB) are activated [190, 192-197]. Known for their ability to modulate the development of various organs and control cell proliferation [198], the notch activated genes and pathways have been reported to drive tumor control through the expansion of CSCs [198-202]. This associated role in selfrenewal function of malignant breast cancers CSCs [198], combined with the fact that Notch inhibitors can kill breast cancer cells *in vitro* and *in vivo*, may partially explain why Notch expression and activation has been associated with a poor prognosis in mammary carcinomas [203-205]. In fact, research findings in breast cancer have presented compelling reasons to target Notch as a therapeutic target in solid tumors. In addition to its ability to regulate survival and proliferation in bulk cancer cells [205] and CSCs [206-209], notch plays a pro-angiogenic role in tumor endothelial cells [210, 211]. Farnie *et al*reported that activated Notch-1, Notch-4, and Notch target Her-1 expression in ductal carcinoma mammospheres *in situ* samples, but not from normal breast tissue [206, 212, 213]. Inhibition of Notch with a gamma-secretase inhibitor (GSI) or a neutralizing Notch-4 antibody has been reported to reduce the ability of ductal carcinoma in situ-derived cells to form mammospheres [207, 214]. Such results suggest that Notch inhibition may have significant therapeutic effects in primary lesions, may be able to preferentially target breast CSCs (responsible for reoccurrence and metastatic disease) and counteract angiogenesis [213]. Cross talk with the NF-κB pathway and Notch1 have been reported in a variety of cell interactions [192, 215-219], including the stimulation of NF-κB promoters[217] and the expression of several NF-κB subunits [192, 215-220].

#### **7.2. Wnt/β-catenin pathway**

The canonical (Wnt/β-catenin) pathway, including Wnt-1, -3A and -8 is likely the best charac‐ terized and traditionally defines Wnt signaling, however other pathways have been described including a non-canonical (planar cell polarity) pathway (including Wnt-5A, - 11) and the Wnt/ Ca2+ pathway (protein kinase A pathway) [221-224]. Although it has been more than 25 years since the discovery of the Wnt gene, its structure remains unknown and signaling pathways are not well defined, especially those independent of β-catenin. Perhaps this challenge can be somewhat explained by the recent discovery that differences in cell signaling outcomes may be attributable to precise pairings of Wnt ligands with analogous cellular receptors [225]. For example, if we consider that the mammalian genome codes for 19 Wnt proteins and 10 Fzd receptors, there are potentially 190 Wnt/Fzd pairing combinations [226]. Although all of these ligand/receptor pairings have not been unveiled, we already know that the Wnt/β-catenin pathway has been established for its ability to alter cell proliferation, migration, apoptosis, differentiation and stem cell self-renewal [224, 227-230]. The essential mediator of the canonical pathway is β-catenin, and its two known distinct functions are based on cell specific locations. Accumulation of β-catenin within the cytoplasm leads to activation of Wnt target genes such as c-Jun, c-Myc, fibronectin and cyclin D1 [186, 231-236]. Prior to nuclear translocation, βcatenin operates in the membrane to maintain cell–cell adhesion via cooperation with the epithelial cell–cell adhesion protein E-cadherin [223]. The Wnt signaling pathway is activated via the binding of ligands to transmembrane receptors encoded by the Frizzled (Fzd) gene family and in conjunction with co-receptors, such as low-density lipoprotein receptors (protein 5 and 6) [237] This Wnt-Fzd interaction results in dephosphorylation, accompanied by decreased levels of degradation and causes the accumulation of β-catenin in the nucleus [231]. In the absence of Wnt signaling, β-catenin is quickly degraded in the cytoplasm. Without Wnt signaling, phosphorylation of adenomatous polyposis coli (APC) [238] via a cytoplasmic destruction complex results in ubiquitination of β-catenin which is then prone to proteasomal degradation [231]. Additionally, nuclear levels of β-catenin are lessened by their interaction with APC and Axin, both known for their function in transporting β-catenin back to the cytoplasm. In the nucleus, transcriptional corepressors interact with DNA-binding T-cell factor/lymphoid-enhancer factor (Tcf/Lef) proteins, such as Groucho/TLE, and are enabled to block target-gene expression when β-catenin is held at low levels. [239-242]. Wnt binding to the Fzd or low-density lipoprotein receptor protein-membrane receptors results in the accumulation and stabilization of translocated (from cytoplasm to nucleus) β-catenin [237]. Inhibition of such interactions has been noted by secreted Fzd-related proteins, Dickkopfs, and Wnt inhibitory factor-1 (WIF-1) [243, 244].

and Hedgehog (Hh) have been identified in human mammary cancer [124, 185, 186]. Addi‐ tionally, evidence has mounted to strengthen the link between nuclear factor kappa-B (NFκB), stem cells and breast cancer as elegantly reviewed in a recent paper by Shostak and Chariot

360 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

Four Notch proteins, namely Notch-1 to Notch-4, are expressed as transmembrane receptors in a variety of stem/progenitor cells [188, 189]. The binding of specific surface-bound ligands are responsible for triggering cleavage events at the Notch proteins by ADAM (A Disintegrin and metalloproteinase domain-containing protein) protease family and γ-secretase [188-191] causing the intracellular domain of Notch to be released and translocate to the nucleus. Once in the nucleus, downstream target genes (including c-Myc, cyclin D1,p21, NF-κB) are activated [190, 192-197]. Known for their ability to modulate the development of various organs and control cell proliferation [198], the notch activated genes and pathways have been reported to drive tumor control through the expansion of CSCs [198-202]. This associated role in selfrenewal function of malignant breast cancers CSCs [198], combined with the fact that Notch inhibitors can kill breast cancer cells *in vitro* and *in vivo*, may partially explain why Notch expression and activation has been associated with a poor prognosis in mammary carcinomas [203-205]. In fact, research findings in breast cancer have presented compelling reasons to target Notch as a therapeutic target in solid tumors. In addition to its ability to regulate survival and proliferation in bulk cancer cells [205] and CSCs [206-209], notch plays a pro-angiogenic role in tumor endothelial cells [210, 211]. Farnie *et al*reported that activated Notch-1, Notch-4, and Notch target Her-1 expression in ductal carcinoma mammospheres *in situ* samples, but not from normal breast tissue [206, 212, 213]. Inhibition of Notch with a gamma-secretase inhibitor (GSI) or a neutralizing Notch-4 antibody has been reported to reduce the ability of ductal carcinoma in situ-derived cells to form mammospheres [207, 214]. Such results suggest that Notch inhibition may have significant therapeutic effects in primary lesions, may be able to preferentially target breast CSCs (responsible for reoccurrence and metastatic disease) and counteract angiogenesis [213]. Cross talk with the NF-κB pathway and Notch1 have been reported in a variety of cell interactions [192, 215-219], including the stimulation of NF-κB

promoters[217] and the expression of several NF-κB subunits [192, 215-220].

The canonical (Wnt/β-catenin) pathway, including Wnt-1, -3A and -8 is likely the best charac‐ terized and traditionally defines Wnt signaling, however other pathways have been described including a non-canonical (planar cell polarity) pathway (including Wnt-5A, - 11) and the Wnt/ Ca2+ pathway (protein kinase A pathway) [221-224]. Although it has been more than 25 years since the discovery of the Wnt gene, its structure remains unknown and signaling pathways are not well defined, especially those independent of β-catenin. Perhaps this challenge can be somewhat explained by the recent discovery that differences in cell signaling outcomes may be attributable to precise pairings of Wnt ligands with analogous cellular receptors [225]. For example, if we consider that the mammalian genome codes for 19 Wnt proteins and 10 Fzd

(2011) [187].

**7.1. Notch pathway**

**7.2. Wnt/β-catenin pathway**

Since the initial observation that Wnt overexpression results in malignant transformation of mouse mammary tissue [245], aberrant regulation of the Wnt signaling pathway has emerged as a prevalent theme and continues to develop as a fundamental mechanism in broad cancer biology [246]. While Wnt pathway mutations (genetic and epigenetic) are rare in mammary carcinoma, overactive Wnt signaling has been noted in the majority of breast cancers, includ‐ ing rare classes (i.e.:triple -ve type)via severalpotentialmechanisms [145, 233, 247-256]. Several studies to date have indicated that the expression of both Wnt receptors and their ligands are characteristic of breast cancer and furthermore certain receptors and ligands may be breast cancer type specific. In 2004, Bafico *et al*. reported autocrine Wnt signaling in a panel of breast cancer cell lines, including MDA-MΒ‐231, which were identified by the presence of unstabi‐ lizedβ‐cateninandthensubsequentlyreduceduponexpressionorbythe additionofthe soluble Wnt inhibitors sFRP1 or DKK1 [257]. The expression of the Wnt receptor FZD7 is characteris‐ tic of certain rare types of breast cancer[258]. Additionally, the knockdown of FZD7 in cell lines representative of triple -ve breast cancer reduced the expression of Wnt target genes, inhibit‐ ed tumorigenesis *in vitro* and greatly retarded the capacity oftheMDA-MD-231 cell line to form tumors inmice[246,259].WithrespecttoWntligands, secretedfrizzledrelatedprotein(sFRP)-1, an effective competitor and binding site with FZD receptors for Wnt ligands, has been shown to be ectopically expressed in the MDA-MΒ‐231 cell line [260]. This same study showed that the sFRP1 expressing cells struggled to form tumors upon inoculation into the mammary fat pads of mice and their propensity to metastasize to lung was greatly impaired [260].

Specific to the maintenance of CSCs, Wnt/β-catenin signaling is implicated in many cancers [223, 224, 261-268], including breast cancer [269]. For example, radiation resistance of mouse mammary stem/progenitor cells has been correlated with overexpression of β-catenin in the stem cell survival pathway [266]. Additionally, overexpression of Wnt/β-catenin signaling was reported to promote expansion of the hepatic progenitor cell population in animal studies [267] and the elimination of β-catenin abrogates chemoresistant cell populations endowed with progenitor-like features [57]. Of great interest is the link between Wnt/β-catenin and PI3K (phosphoinositide 3 kinase) /Akt (protein kinase B) pathway as established by several studies. Korkaya *et al*. demonstrated that PI3K/Akt pathway is important in regulating the mammary stem/progenitor cells by promoting β-catenin downstream events through phosphorylation of GSK3β [60, 189]. Other studies have revealed the ability of activated Akt, such as phospho-Akt Ser473 to phosphorylate Ser9 on GSK3β, thereby decreasing the activity of GSK3β, and potentially stabilizing β-catenin [270-272].

In summary, the proof of concept for inhibiting Wnt signaling in cancer is in place. Furthermore there is an increasing amount of evidence to support a role for Wnt signaling in breast cancer; thus, a target has been created for future studies. Specific to breast cancer, the emphasis on target development ranges from antagonizing Wnt ligand secretion or binding to promote βcatenin degradation to specifically blocking β-catenin-mediated transcriptional activity [222]. Nonetheless, as noted several times throughout this chapter, the cooperation of Wnt pathway with other signaling pathways in cancer is an important consideration. Aside from the challenge of determining the most efficacious way to inhibit Wnt related factors, possible safety concerns should be considered; another compelling reason to explore specific targets in the Wnt pathways for all breast cancer sub-types.

#### **7.3. Hh pathway**

A crucial mediator of normal tissue development, with recent indications as a regulator of tumor-related vascular formation and function [273], the Hh signaling pathway in cancer is activated by ligand independent mutations in the pathway or through Hh overexpression (ligand-dependent) [189, 274, 275]. In the absence of Hh ligands, (Sonic Hh, Desert Hh and Indian Hh), their transmembrane receptor Patched (Ptch) associates with and blocks the Gprotein-coupled phosphoprotein receptor Smoothened (Smo) and is only released when secreted Hh ligands bind to Ptch [189, 276, 277]. This binding triggers the dissociation of glioma-associated (Gli) family of zinc finger transcription factors. The three Gli proteins found in vertebrates include Gli1 and Gli2 (thought to activate Hh target genes) and Gli3 (known to act primarily as a repressor) which lead to the transcription of an assortment of genes including cyclin D, cyclin E, myc and elements of EGF pathway effectors through complex interactions with Costal2 (Cos2), Fused (Fu) and Suppressor of Fu (SuFu) [276-278]. Somatic mutations which activate Hh pathway have been implicated in a variety of human malignancies[278] including basal cell carcinomas, pancreatic cancer, medulloblastomas, leukemia, gastrointes‐ tinal, lung, ovarian, breast and prostate cancers [274, 275, 279]. Both *in vitro* and mouse model systems have demonstrated that the Hh signaling pathway plays a crucial role in regulating self-renewal of normal and malignant human mammary stem cells [51, 189]. Hh pathway inhibition has been shown to result in tumor growth inhibition mediated through the stromal microenvironment; as demonstrated in a xenograft model using a tumor and stromal cell coinjection procedure, and consistent with a paracrine signaling mechanism [280]. Although data describing the genetic alteration and the modulation of the expression pattern of Hh pathway components in mammary gland are limited, possible indications for the Hh pathway in development and maintenance of mammary cancer have been proposed [281]. However, a more significant role of Hh signaling has been revealed in prostate cancer studies, demon‐ strating that autocrine Hh signaling by tumor cells is a requirement for proliferation, viability and invasive behavior [282]. Additionally, the association of accelerated prostate cancer growth and progression with increased Hh signaling has been reported [283]. The Hh signaling pathway has been demonstrated as a critical pathway involved in stem cell self-renewal [276] including the essential role of Hh-Gli signaling in controlling the self-renewal behavior of human glioma CSCs and tumorigenicity [189, 284]. Known for its central role in the control of proliferation and differentiation of both embryonic stem cells and adult stem cells, aberrant activation of Hh signaling could be involved in the generation of CSCs and the development of cancer [278, 285]. In this regard, the development of Hh inhibitors may be a solution in the treatment of human cancers, including prevention of tumor progression. Essential similarities have been noted between Wnt and Hh signaling pathways [286] and their key roles in the physiological and pathological development of both embryonic and stem cells [278] [278]gives rise to the fact that crosstalk exists between the two. Signaling for both are activated by Gprotein-coupled receptors [287, 288] and prevents phosphorylation-dependent proteolysis of key effectors (*Cubitus interruptus* or β-catenin) responsible for the conversion of a DNA-binding protein from a repressor to an activator of transcription [278, 289]. Considering the progression model of many cancers, specifically metastasis to bone, it is interesting to note that Wnt signaling has been reported to be downstream of Hh signaling, participating in bone devel‐ opment [278, 290]. Further proof proposing that Wnt signaling is downstream of Hh includes the ability of activated Gli1 to stimulate the transcription of Wnt ligands [276, 278]. It has been noted that molecules involved in Wnt signaling (i.e: GSK-3β) also play a regulatory role in Hh signaling [278, 286]. Furthermore, canonical Wnt/β-catenin signaling is required for the pathological response to oncogenic Hh signaling [278, 291].

#### **7.4. NFĸB Pathway**

sFRP1 expressing cells struggled to form tumors upon inoculation into the mammary fat pads

Specific to the maintenance of CSCs, Wnt/β-catenin signaling is implicated in many cancers [223, 224, 261-268], including breast cancer [269]. For example, radiation resistance of mouse mammary stem/progenitor cells has been correlated with overexpression of β-catenin in the stem cell survival pathway [266]. Additionally, overexpression of Wnt/β-catenin signaling was reported to promote expansion of the hepatic progenitor cell population in animal studies [267] and the elimination of β-catenin abrogates chemoresistant cell populations endowed with progenitor-like features [57]. Of great interest is the link between Wnt/β-catenin and PI3K (phosphoinositide 3 kinase) /Akt (protein kinase B) pathway as established by several studies. Korkaya *et al*. demonstrated that PI3K/Akt pathway is important in regulating the mammary stem/progenitor cells by promoting β-catenin downstream events through phosphorylation of GSK3β [60, 189]. Other studies have revealed the ability of activated Akt, such as phospho-Akt Ser473 to phosphorylate Ser9 on GSK3β, thereby decreasing the activity of GSK3β, and

In summary, the proof of concept for inhibiting Wnt signaling in cancer is in place. Furthermore there is an increasing amount of evidence to support a role for Wnt signaling in breast cancer; thus, a target has been created for future studies. Specific to breast cancer, the emphasis on target development ranges from antagonizing Wnt ligand secretion or binding to promote βcatenin degradation to specifically blocking β-catenin-mediated transcriptional activity [222]. Nonetheless, as noted several times throughout this chapter, the cooperation of Wnt pathway with other signaling pathways in cancer is an important consideration. Aside from the challenge of determining the most efficacious way to inhibit Wnt related factors, possible safety concerns should be considered; another compelling reason to explore specific targets in the

A crucial mediator of normal tissue development, with recent indications as a regulator of tumor-related vascular formation and function [273], the Hh signaling pathway in cancer is activated by ligand independent mutations in the pathway or through Hh overexpression (ligand-dependent) [189, 274, 275]. In the absence of Hh ligands, (Sonic Hh, Desert Hh and Indian Hh), their transmembrane receptor Patched (Ptch) associates with and blocks the Gprotein-coupled phosphoprotein receptor Smoothened (Smo) and is only released when secreted Hh ligands bind to Ptch [189, 276, 277]. This binding triggers the dissociation of glioma-associated (Gli) family of zinc finger transcription factors. The three Gli proteins found in vertebrates include Gli1 and Gli2 (thought to activate Hh target genes) and Gli3 (known to act primarily as a repressor) which lead to the transcription of an assortment of genes including cyclin D, cyclin E, myc and elements of EGF pathway effectors through complex interactions with Costal2 (Cos2), Fused (Fu) and Suppressor of Fu (SuFu) [276-278]. Somatic mutations which activate Hh pathway have been implicated in a variety of human malignancies[278] including basal cell carcinomas, pancreatic cancer, medulloblastomas, leukemia, gastrointes‐ tinal, lung, ovarian, breast and prostate cancers [274, 275, 279]. Both *in vitro* and mouse model

of mice and their propensity to metastasize to lung was greatly impaired [260].

362 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

potentially stabilizing β-catenin [270-272].

Wnt pathways for all breast cancer sub-types.

**7.3. Hh pathway**

Along with their hallmark roles in cell survival, proliferation, inflammation and immunity, the NF-κB family of transcription factors are often constitutively expressed in breast cancer tumors [292]. Early studies on NF-κB pathway determined its key role in mammary epithelial proliferation, architecture and branching during early post-natal development [293, 294]. However, independent of its effects on mammary development, evidence exists to suggest that NF-κB regulates breast tumor progression [293, 294]. Constitutive activation of NF-κB in several breast tumor cell lines has been shown to profoundly affect the initiation and progres‐ sion of breast cancer [295]. NF-κB is also required for the induction and maintenance of the EMT a process that critically controls breast cancer progression [296, 297]. Additionally, it is evident that NF-κB mostly acts in specific breast cancer sub-types, namely estrogen receptor (ER)-ve and ErbB2+ve tumors [298, 299] and has been implicated in stem cell expansion in breast cancer studies [187].

Activation of NF-κB results in the constant nuclear localization of proteins including p50, p52, p65, cRel and RelB which subsequently up-regulate anti-apoptotic proteins causing an imbalance between normal cell growth and apoptotic cell death [300]. NF-κB-activation occurs mainly through two well characterized pathways, namely the canonical (classical) and the noncanonical (alternative). Both pathways systematically work in a similar fashion in that they are reliant on signal-induced phosphorylation and degradation of an inhibitory molecule to release and transport nuclear NF-κB proteins. However, they differ in the types of trigger signals, activated kinases, inhibitory molecules and NF-κB proteins utilized in each system. In addition to each of these aforementioned pathways, other NF-κB activating pathways exist and have been indicated in the initiation and progression of breast cancer, however we will not discuss these fully in this chapter other than in the context that they appear in the described and relevant research experiments.

Specifically, the canonical pathway involves translocation of a p50/p65 heterodimer to prompt the expression of genes intricated in cell proliferation as well as their survival, inflammatory properties and role in innate immunity [292]. This process occurs through a transforming growth factor beta activated kinase-1 (TAK1)-dependent pathway and is normally dependent on members of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) or IL-1β and other pro-inflammatory cytokines to degrade the inhibitor (IκBα) by the NF-κB essential modulator ((NEMO)/IκB kinase (IKK))γ-containing IKK complex [292]. In 2004, Biswas *et al.* published results regarding the activation of NF-κB in human breast tumors and in carcinoma cell lines indicating that the canonical pathway contributes to tumor development [298]. The resulting highlight of this experiment gave rise to activated NF-κB as a therapeutic target for distinctive subclasses of ER-ve breast cancers [298]. Specifically, the (NEMO)-binding domain (NBD) peptide (a selective inhibitor of IKK) blocked heregulin-mediated activation of NF-κB and cell proliferation while inducing apoptosis on proliferating cells substantiating the hypothesis that certain breast cancer cells rely on NF-κB for aberrant cell proliferation and simultaneously avoid apoptosis [298]. More recently, Connelly *et al*. [2011] showed, via genetic approaches, that the canonical NF-κB-activating pathway is inhibited in defined frames during polyoma middle T oncogene (PyVT) tumorigenesis and that interruption of this pathway in the mammary epithelium increases the latency of tumors and decreases tumor burden [301].

The non-canonical pathway, considered to be critical in adaptive immunity, is similar to the canonical cascade as it also relies on an IKKα heterodimer, but not on NEMO/IKKγ [302]. Prior to nuclear shuttling of 52/RelB dimers, the inhibitory molecule p100 is partially degraded through an NF-κB-inducing kinase (NIK)-dependent pathway [292]. Early studies revealed the enhanced expression of the NF-κB protein p52 in breast cancer samples giving rise to the involvement of the non-canonical pathway [303],[304]. The NF-κB protein RelB is increased in ERα-ve breast cancer cells and is required for the maintenance of mesenchymal ERα-ve breast cancer cells partially through the transcriptional induction of BCL2 [305]. Furthermore, RelB/ p52 complexes have since been implicated in mammary carcinogenesis. For example, mouse mammary tumors induced by 7,12-dimethylbenz(a)anthracene treatment have been shown to increase RelB/p52 activity and the inhibition of RelB in breast cancer cells repressed cyclin D1 and c-Myc levels and growth in soft agar [306]. Perhaps the most conclusive proof of noncanonical NF-κB-activating pathway involvement occurred in studies employing a novel transgenic mouse model to consider the role of involved mediators (downstream of p100/p52] in both mammary development and tumorigenesis [307]. The results of this study indicated an increase in p100/p52 expression in tumors from mice expressing PyVT in the mammary gland, [307] with no change of nuclear p65 detected; an indication that the observation was limited to a deregulated non-canonical NF-κB-activating pathway [307].

#### *7.4.1. NF-κB and breast cancer stem cell renewal*

evident that NF-κB mostly acts in specific breast cancer sub-types, namely estrogen receptor (ER)-ve and ErbB2+ve tumors [298, 299] and has been implicated in stem cell expansion in

364 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

Activation of NF-κB results in the constant nuclear localization of proteins including p50, p52, p65, cRel and RelB which subsequently up-regulate anti-apoptotic proteins causing an imbalance between normal cell growth and apoptotic cell death [300]. NF-κB-activation occurs mainly through two well characterized pathways, namely the canonical (classical) and the noncanonical (alternative). Both pathways systematically work in a similar fashion in that they are reliant on signal-induced phosphorylation and degradation of an inhibitory molecule to release and transport nuclear NF-κB proteins. However, they differ in the types of trigger signals, activated kinases, inhibitory molecules and NF-κB proteins utilized in each system. In addition to each of these aforementioned pathways, other NF-κB activating pathways exist and have been indicated in the initiation and progression of breast cancer, however we will not discuss these fully in this chapter other than in the context that they appear in the described

Specifically, the canonical pathway involves translocation of a p50/p65 heterodimer to prompt the expression of genes intricated in cell proliferation as well as their survival, inflammatory properties and role in innate immunity [292]. This process occurs through a transforming growth factor beta activated kinase-1 (TAK1)-dependent pathway and is normally dependent on members of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNFα) or IL-1β and other pro-inflammatory cytokines to degrade the inhibitor (IκBα) by the NF-κB essential modulator ((NEMO)/IκB kinase (IKK))γ-containing IKK complex [292]. In 2004, Biswas *et al.* published results regarding the activation of NF-κB in human breast tumors and in carcinoma cell lines indicating that the canonical pathway contributes to tumor development [298]. The resulting highlight of this experiment gave rise to activated NF-κB as a therapeutic target for distinctive subclasses of ER-ve breast cancers [298]. Specifically, the (NEMO)-binding domain (NBD) peptide (a selective inhibitor of IKK) blocked heregulin-mediated activation of NF-κB and cell proliferation while inducing apoptosis on proliferating cells substantiating the hypothesis that certain breast cancer cells rely on NF-κB for aberrant cell proliferation and simultaneously avoid apoptosis [298]. More recently, Connelly *et al*. [2011] showed, via genetic approaches, that the canonical NF-κB-activating pathway is inhibited in defined frames during polyoma middle T oncogene (PyVT) tumorigenesis and that interruption of this pathway in the mammary epithelium increases the latency of tumors and decreases tumor burden [301].

The non-canonical pathway, considered to be critical in adaptive immunity, is similar to the canonical cascade as it also relies on an IKKα heterodimer, but not on NEMO/IKKγ [302]. Prior to nuclear shuttling of 52/RelB dimers, the inhibitory molecule p100 is partially degraded through an NF-κB-inducing kinase (NIK)-dependent pathway [292]. Early studies revealed the enhanced expression of the NF-κB protein p52 in breast cancer samples giving rise to the involvement of the non-canonical pathway [303],[304]. The NF-κB protein RelB is increased in ERα-ve breast cancer cells and is required for the maintenance of mesenchymal ERα-ve breast cancer cells partially through the transcriptional induction of BCL2 [305]. Furthermore, RelB/ p52 complexes have since been implicated in mammary carcinogenesis. For example, mouse

breast cancer studies [187].

and relevant research experiments.

Recently, studies have strengthened the association between stem cells, breast cancer and NFκB. Such have been captured in a review by Shostak and Chariot highlighting experiments to date complemented by a compelling rationale for targeting NF-κB and other developmental pathways involved in the self-renewal of normal stem cells [187]. The involvement of NF-κB in various signaling cascades has proven critical in several studies involving breast cancer stem cell expansion.

Cao *et al*. [2007] showed that IKKα is both a regulator of mammary epithelial proliferation and a contributor to ErbB2-induced oncogenesis [308]. Specifically, breast cancer cells from IKKα (AA/AA) knock-in mice (whereby IKKα activation is disrupted) crossed with the Her2 murine breast cancer model, exhibited diminished self-renewal capacity and resulted in the inability to establish secondary tumors [308]. Breast cancers that generate primary, as opposed to secondary mammospheres such as seen in mice used in these experiments, suggests that IKKα is likewise required for the self-renewal of tumor-initiating cells from the Her2 breast cancer model [187]. Additionally, mutated IKKα slowed tumor development following exposure to 7,12-dimethylbenzaanthracene or the MMTV-c-neu (ErbB2/Her2) transgene; however there was no effect on MMTV-v-Ha-ras-induced cancer despite the fact that both of these oncogenes rely on cyclin D1 [308]. In this same series of studies, carcinoma cells from another mouse model (IKKα(AA/AA)/MMTV-c-neu) underwent premature senescence when cultured under conditions used for propagation of mammary gland stem cells. Altogether, these mouse models of breast cancer show that IKKα seems to act as a central protein in the activation of NF-κB during breast cancer stem cell self-renewal [308]. Therefore, the researchers concluded that IKKα may represent a novel and specific target for treatment of ErbB2+ve breast cancer.

While NF-κB appears to be activated in luminal progenitor cells during differentiation of mammary colony-forming cells [309], the mammary stem-like basally located cells are devoid of NF-κB activity [309, 310]. Taken together, these studies suggest that only the canonical NFκB pathway is active in normal luminal progenitor cells before transformation and is re‐ quired for the formation of mammary luminal-type epithelial neoplasias [309]; a reminder of the importance of understanding the cellular etiology underpinning breast tumor heterogene‐ ity [310].

Another interesting role for NF-κB signaling involves the link between inflammation and cancer. While the mechanism linking inflammation and cancer has yet to be explained, we do know that the inflammatory cytokine, interleukin(IL)-6 is up-regulated in epithelial cancers, including breast cancer [311]. We also know that NF-κB regulates the expression of antiapoptotic genes and activates different pro-inflammatory cytokines and chemokines, includ‐ ing IL-6 [312, 313]. Further clarity on the interactions of NF-κB signaling, inflammation and cancer has been gained through a study showing that the temporary activation of Src onco‐ protein mediates an epigenetic event whereby immortalized breast cells are stably transformed to a cell lines that represent self-renewing mammospheres containing cancer stem cells [313]. The inflammatory response triggered by the activation of Src and further downstream signaling which inhibits IL-6 expression is mediated by NF-κB [313]. It has been shown that the transformation of cells utilized within this experiment occurs via a positive feedback loop whereby IL-6 mediated STAT3 transcription factor stimulation activates NF-κB [313]. These authors have demonstrated that Src activation triggers a rapid inflammatory response mediated by NF-κB that is critical for cellular transformation. While this study defines Src's role as an oncogenic kinase promoting the expansion of breast cancer stem cells, it also demonstrated the critical involvement of NF-κB in the process [313].

It is known that the onset of progestin-driven breast cancer is affected by the deletion of IKKα in mammary-gland epithelial cells [314]. Such studies are relevant in breast cancers as they consider the importance of associated risk factors between hormone replacement therapy (i.e.: progesterones or synthetic derivatives) and the increased risk of incident of fatal breast cancer [314]. The expression of both receptor activator of NF-κB (RANK) and RANK ligand (RANKL) have been observed in primary breast cancers in humans and breast cancer cell lines [315]. Studies to date indicate that the RANKL/ RANK system is mediated in part by IKK-α– NF-κB signaling and controls the incidence and onset of progestin-driven breast cancer; more specifically a loss of RANK expression significantly impairs the self-renewal capacity of cancer stem cells [314, 316]. Thus, because of the link between the RANKL/RANK system and progestin-driven epithelial carcinogenesis, RANKL inhibition could be considered as a novel approach to the prevention and/or treatment of breast cancer [314].

More recently, a model of Her2-dependent tumorigenesis indicated that breast cancer stem cell renewal is regulated by epithelial NF-κB through a reduction in the expression of key embryonic stem cell regulators, namely Sox2 and Nanog [317]. Specifically, NF-κB was required for both proliferation and colony formation of Her2-derived murine mammary tumor cell lines [317]. Additionally, the rate of initiation of Her2 tumors was governed by NF-κB [317].

#### **7.5. Summarizing pathway interruptions/targets**

In many human breast cancers, all three developmental pathways (Wnt, Notch and Hh) appear to be deregulated and control the self-renewal of normal stem cells from a molecular perspec‐ tive [186]. Additionally, the involvement of NF-κB has emerged as another involved pathway in breast cancer based on what we know about Her2, a membrane bound receptor tyrosine kinase. Her 2 is overexpressed in 30% of breast cancers and critically controls the cancer stemcell population [318]. Since Her2 activates NF-κB through the canonical pathway [319] [27], the hypothesis exists that the NF-κB pathway may be involved in the biology of breast cancer stem cells. It is also obvious that these interrupted pathways, and likely others unknown at this time, are responsible for certain stages of cancer progression or cancer cell aggression. If so, then we are in agreement with others who have noted that the development and identifi‐ cation of selective inhibitors of specific signaling pathways is an attractive approach for the prevention of tumor progression and/or treatment of cancers [278]. However, we have also mentioned several examples of cross-talk between the components of different cell signaling pathways. This concept then introduces the task of targeting multiple pathways in an effort to prevent progression and metastasis of cancers. Alternatively, and based on the premise that certain pathways operate downstream of others, it would be more reasonable to focus on inhibitors of overarching pathways, such as NF-κB. In summary, such oncogenic pathway signatures are fundamental in natural product testing. Therapeutic approaches involving natural products may provide a link between pathway deregulation and therapeutic sensitiv‐ ity indicating an opportunity for the development of target compound(s).

Another interesting role for NF-κB signaling involves the link between inflammation and cancer. While the mechanism linking inflammation and cancer has yet to be explained, we do know that the inflammatory cytokine, interleukin(IL)-6 is up-regulated in epithelial cancers, including breast cancer [311]. We also know that NF-κB regulates the expression of antiapoptotic genes and activates different pro-inflammatory cytokines and chemokines, includ‐ ing IL-6 [312, 313]. Further clarity on the interactions of NF-κB signaling, inflammation and cancer has been gained through a study showing that the temporary activation of Src onco‐ protein mediates an epigenetic event whereby immortalized breast cells are stably transformed to a cell lines that represent self-renewing mammospheres containing cancer stem cells [313]. The inflammatory response triggered by the activation of Src and further downstream signaling which inhibits IL-6 expression is mediated by NF-κB [313]. It has been shown that the transformation of cells utilized within this experiment occurs via a positive feedback loop whereby IL-6 mediated STAT3 transcription factor stimulation activates NF-κB [313]. These authors have demonstrated that Src activation triggers a rapid inflammatory response mediated by NF-κB that is critical for cellular transformation. While this study defines Src's role as an oncogenic kinase promoting the expansion of breast cancer stem cells, it also

It is known that the onset of progestin-driven breast cancer is affected by the deletion of IKKα in mammary-gland epithelial cells [314]. Such studies are relevant in breast cancers as they consider the importance of associated risk factors between hormone replacement therapy (i.e.: progesterones or synthetic derivatives) and the increased risk of incident of fatal breast cancer [314]. The expression of both receptor activator of NF-κB (RANK) and RANK ligand (RANKL) have been observed in primary breast cancers in humans and breast cancer cell lines [315]. Studies to date indicate that the RANKL/ RANK system is mediated in part by IKK-α– NF-κB signaling and controls the incidence and onset of progestin-driven breast cancer; more specifically a loss of RANK expression significantly impairs the self-renewal capacity of cancer stem cells [314, 316]. Thus, because of the link between the RANKL/RANK system and progestin-driven epithelial carcinogenesis, RANKL inhibition could be considered as a novel

More recently, a model of Her2-dependent tumorigenesis indicated that breast cancer stem cell renewal is regulated by epithelial NF-κB through a reduction in the expression of key embryonic stem cell regulators, namely Sox2 and Nanog [317]. Specifically, NF-κB was required for both proliferation and colony formation of Her2-derived murine mammary tumor cell lines [317]. Additionally, the rate of initiation of Her2 tumors was governed by NF-κB [317].

In many human breast cancers, all three developmental pathways (Wnt, Notch and Hh) appear to be deregulated and control the self-renewal of normal stem cells from a molecular perspec‐ tive [186]. Additionally, the involvement of NF-κB has emerged as another involved pathway in breast cancer based on what we know about Her2, a membrane bound receptor tyrosine kinase. Her 2 is overexpressed in 30% of breast cancers and critically controls the cancer stemcell population [318]. Since Her2 activates NF-κB through the canonical pathway [319] [27],

demonstrated the critical involvement of NF-κB in the process [313].

366 Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century

approach to the prevention and/or treatment of breast cancer [314].

**7.5. Summarizing pathway interruptions/targets**
