Extrinsic cause of resistance

The interaction between the microenvironment and CSC is a dynamic process resulting in continuous remodeling of both.84 EMT plays a crucial role in chemoresistance and aids in cancer metastasis.92Augmented drug efflux, suppressed apoptotic signaling pathways, and slow cellular proliferation are associated with EMT, and this contributes to the resistance of BC cells against anticancer drugs.93Gefitinib or erlotinib, prescribed to BC patients with high EGFR, often relapses. EMT-associated transcription factor Snail is reported to enhance the expression of AXL receptor tyrosine kinase. Signals transduced by AXL allow the BC cells to override the cytostatic effects of EGFR inhibitors. EMT also triggers other processes that enable the BC cells to elude the lethal effect of cytotoxic T cells. Elevated expression of PD-L1 is one such major evasion mechanism employed by BC cells.75 PD-1, an inhibitory immune-checkpoint receptor, expressed by cytotoxic T cells recognizes the PD-L1 on the cancer cells and diminishes their function. Further, enhanced secretion of thrombospondin-1 by mesenchymal cells induces the development of regulatory T cells within the tumor microenvironment that suppresses the cytotoxic T cells.

In addition to chemoresistance, EMT process also equips the BC cells to evade cytotoxic effects of radiation. Radio-resistant BC cells that acquire mesenchymal properties have been reported to be more invasive, attributed to enhanced traction forces and membrane ruffling.94 The intricate program of EMT is not only responsible for the development of BCSC, but deeper understanding in the process would help us to develop novel approaches to target cells that evade conventional therapeutic regimens. Paracrine signals from the Notch/Wnt/Hedgehog pathway influence EMT by cytoskeleton rearrangements which results in mesenchymal-like phenotype.95

BCs have the tendency to recruit mesenchymal cells from the normal breast stroma96 or from the bone marrow.97 For instance, mesenchymal stem cells (MSCs) expressing ALDH1 are selectively recruited to areas of actively dividing tumor, where they interact with BCSCs via cytokine loops of CXCL7 and IL-6.97 These cytokine signaling augments the self-renewal of BCSCs.97 In addition, MSCs have also been shown to protect the BCSCs via recruitment of regulatory T cells.98Immunohistochemical analysis has established the presence of such interacting MSC/BCSC in tumor biopsies of BC patients.97 High ALDH1 expression in BC cells has been shown to be an independent predictor of poor outcome in patients with BC.79 Further, MSCs have the capability to differentiate into adipocytes and tumor-associated fibroblasts, which might also interact with tumor cells and can influence disease progression.99

Gabbiani and Majno were the first who reported the morphological alterations in the stimulated myofibroblasts of dormant tumor- and wound-associated fibroblasts.100 Confirming the observation, in an experimental mouse model, it was demonstrated that acute wounding of the mammary gland by dermal incision augmented BC growth and metastasis.101 While the exact underpinning mechanisms remain elusive, it is believed that paracrine signals from the developing tumors induce epigenetic changes in the neighboring stromal fibroblasts.102 Certainly, the expression profile of cancer-associated fibroblasts (CAFs) is similar to that of wound-associated fibroblasts; this profile has been linked with poor outcome of patients.103,104 A report suggests that transforming growth factor beta (TGF-β; growth factor) may be involved in regulating the epigenetic changes, leading to fibroblast activation.105 In addition, cytokines like CXCL12 (also known as SDF-1) released by BC-associated fibroblasts might help in the proliferation of cancer cells, which expresses CXCR4 (SDF-1 receptor).106The high levels of free SDF-1 in serum has been correlated with poor outcome in BC patients.107,108

Interleukin-6 (IL-6) and IL-8 have been associated with both chronic inflammation and tumor growth.109,110 Many cell types present in the tumor microenvironment including macrophages, immune cells, and mesenchymal cells have been reported to secrete both IL-6 and IL-8.110 In addition, the high levels of both of these cytokines in serum have been related to poor BC patient outcome.111,112 IL-6 has been shown to promote angiogenesis, tumorigenicity, and metastasis.113 In clinics, correlation of high IL-6 serum levels and poor outcome in BC patients justifies the studies aimed to elucidate the role that these cytokines play in tumorigenesis. A report demonstrated that IL-6 is directly involved in BCSC self-renewal, that was mediated by the IL-6 receptor/GP130 complex via STAT3 activation.114 IL-6 has been shown to be a vital element of positive feedback loop that regulates these MSCs and BCSCs.97 Utilizing relative gene expression profiling, it was identified that CXCR1 (IL-8 receptor) was overexpressed on BCSCs and also IL-8 was able to induce the self-renewal of the BCSCs.115 Further, blocking the receptor activity in mouse xenografts significantly reduced the population of BCSCs, resulting in decreased tumorigenicity and metastasis. The production of inflammatory cytokines such as IL-6 and IL-8 is controlled by the NF-κB signaling pathway.116

Hepatocyte growth factor (HGF), released by mammary stromal cells, might also play an important role in developing mammary tumors.117 HGF serves as a co-stimulatory signal to activate the Wnt pathway during colon carcinogenesis;118 however, involvement of similar pathways in breast carcinogenesis is still unknown. Another vital growth factor released by activated fibroblasts includes fibroblast growth factors (FGFs). It was recently reported that estrogen regulates the BCSC population via paracrine signaling cascade involving FGF9.119 Additional factors such as PDGF, IGF, Wnt, Hedgehog ligands, Notch ligands, and matrix metalloproteinases (MMPs) are released in the tumor microenvironment that controls tumor proliferation, invasion, and metastasis.120–125

Endothelial cells are involved in blood vessel formation and might play an important role in developing the tumor microenvironment via direct interaction with tumor cells. Endothelial cells have been reported to be a significant constituent of normal neuronal and hematopoietic stem cell niches.126,127It has been observed that cytokines produced by endothelial cells regulate CSCs.128,129 Remarkably, the tumor vasculature is substantially different from the normal vasculature, as exemplified by the differential expression of almost 1,000 genes between them, including JAK3, MMPs, and FGF receptors.128 Even though several pro-angiogenic factors have been recognized, VEGF is the principal facilitator of this process,130 and because of this, it has become the primary target of many anti-angiogenic therapeutics. Bevacizumab and two small molecule multi-kinase VEGF inhibitors, sunitinib and sorafenib, are currently approved for clinical application. Bevacizumab was approved against metastatic BC as it can prolong the time taken to tumor progression.131 However, more recent studies have suggested discouraging the result that the effect is severely limited and that the combination of bevacizumab and cytotoxic chemotherapy failed to increase the OS of patient.132 These results are corroborated with reports in mouse models that application of anti-angiogenic agents might accelerate BC invasion and metastasis.133,134 By studying mouse model of human BC, a report also suggests that these anti-angiogenic agents increase the CSC pool through tissue hypoxia.135 Anti-angiogenesis drugs might also augment tumor growth by stimulating HGF production from tumor-associated stromal cells.136

New approaches to develop therapeutics against BCSCs

As stated above resistance to conventional therapeutic regime such as radiation and cytotoxic chemotherapy has been the motivation behind the development of specific agents capable of targeting the CSC population. BCSCs show enhanced expression of CD44 and ABC transporters promoting survival of these stem cells. These surviving cells again give rise to tumors that have enhanced chemo-tolerance and metastatic ability resulting in relapse. Here we would discuss some of the current approaches used for targeting BCSC. The promising therapies employed against BCSCs have been summarized in Table 1.

Targeting signaling cascades

There are significant reports of dysregulation of Notch pathway in a substantial fraction of human BCs.137,138 Of the various approaches, one of the most clinically promising candidates is γ-secretase inhibitor. Activation of Notch signaling is regulated by this proteolytic enzyme (γ-secretase), which cleaves Notch receptors and releases the intracellular domain, which in turn acts as a transcription factor and regulates important oncogenic gene functions.139 Therefore, γ-secretase inhibitors were designed for treating BC patients in an early-phase clinical trial. The most severe effect observed has been the gastrointestinal toxicity due to goblet cell hyperplasia, which is an on-target effect of Notch inhibition.140 Although, a moderate dosage along with the administration of high dose of corticosteroids was able to lower the toxicity.141 Combination of γ-secretase inhibitor with taxane chemotherapy has also been clinically investigated in a Phase I trial.141 Other pathway regulating BCSC is the Hedgehog pathway. This pathway has been reported to be active in tumor cells as well as in the tumor stroma.142 Oral Hedgehog inhibitors were clinically tested, and they appear to be fairly nontoxic.143 Phase II clinical studies employing this drug compounds in combination with conventional cytotoxic agents are underway.

Despite the outstanding clinical efficiency of HER2-targeted therapy, almost one third of HER2-positive BC patients do not respond to these agents, and chemoresistance may develop in these patients with chronic exposure. Increasing evidence indicates that resistance may be associated with the activation of other receptor kinases, gain of function mutations of PI3K, loss of PTEN tumor suppressor gene, or truncation of the extracellular domain of HER2.144 These mutations cause aberrant activation of the downstream PI3K/Akt/mTOR pathway and are generally correlated with poor prognosis after conventional trastuzumab therapy.144 Confirmatory evidence has recently shown that the PI3K/Akt/mTOR pathway plays a significant role in regulating BCSC pool. This ensues via Akt activation of the Wnt pathway through phosphorylation of GSK/3β and direct phosphorylation of β-catenin on serine552 amino acid which results in its nuclear transport.145 This observation suggests that suppressing Akt that is downstream of HER2 signaling might efficiently target BCSCs in HER2-resistant tumors. Indeed, perifosine (Akt inhibitor) has demonstrated promising prospect by effectively targeting the BCSC pool in breast tumor xenografts.145 Encouraged by the abovementioned observations, a spectrum of PI3K and Akt selective inhibitors are being clinically investigated, providing us the direct assessment of the effects of these agents in controlling the stem cell population.

Targeting tumor microenvironment

The role of cytokine signaling in maintaining and promoting CSCs is well-documented. Among the cytokines, IL-6 and IL-8 play an important role in the maintenance of BCSC population. These two cytokines promote the inflammatory cascade via NF-κB pathway leading to chronic inflammation and augmentation of tumorigenesis. Interestingly, anti-inflammatory agents such as statins show a decrease in BC risk.146 Statins lower the levels of pro-inflammatory cytokines, as revealed by lowered CRP levels.147 A recent report has demonstrated that antibodies against the CXCR1 (IL-8 receptor) or repertaxin (small molecule CXCR1/CXCR2 inhibitor) has the potential to target BCSC in mouse xenograft models impeding tumor growth and metastasis.145 Repertaxin was initially developed to avert graft rejection and has shown to have promising effect in Phase I trials. Repertaxin was reported to mediate stem cell death in bulk cellular population through bystander effect involving release of Fas cell surface death receptor (FAS) ligand. CXCR1 inhibits FOXO3A localization and FAS ligand expression through AKT signaling. Treatment with repertaxin suppressed AKT, resulting in nuclear FOXO3A and FAS ligand expression. Conventional chemotherapeutics are also known to cause cell death via a bystander effect through FAS ligand, but that in turn induces IL-8 which protects BCSC from FAS ligand. This suggested that repertaxin might cause blockade of this effect and efficiently kill the BCSC population. Repertaxin also suppressed the BCSC population in vitro as well as in tumor xenografts. As a monotherapy, repertaxin had a nonsignificant effect on tumor growth, but drastically reduced tumor volume when employed in combination with docetaxel. Further, repertaxin was able to reduce metastatic lesions and secondary tumor formation. These promising results show that repertaxin can sensitize BCSC to bystander effect via FAS ligand and that CXCR1 blocking might represent a novel approach to targeting and eliminating breast CSCs. Further, mAbs targeting IL-6 or its receptor are currently being assessed in clinical trials for multiple myeloma.148

The downregulation of caveolin-1 (CAV1) in CAFs is a well-studied biomarker which is associated with oncogenic transformation. It has been observed that inhibition of CAV1 in CAFs resulted in hyper-proliferative phenotype of BC cells. The replacement of CAV1 with CAV1 mimetic eliminated the proliferative behavior of the cells.149 It was also reported that CAFs and MSCs sensitized MCF7 cells to the RAD001 (an mTOR inhibitor) and augmented the cytotoxic effect of RAF265 (an RAF inhibitor) on MDA-MB-231 cells through the inhibition of ERK1/2 phosphorylation.150 However, both CAFs and MSCs had no significant effect on the response to TKI258 (a PDGFR/FGFR/VEGFR inhibitor) in BC cell lines.150 This demonstrates that CAFs may not be involved in all the mechanisms of drug resistance, but heterogeneity of CAFs should be taken into account during drug response.

Tumor and adjoining stromal cells are known to secrete CCL2, which is an essential chemoattractant for macrophages. CCL2 and its receptor, CCR2, are involved in monocyte recruitment onto the tumor periphery. Lu et al observed that overexpression of CCL2 promotes both bone and lung metastases in BC. Targeting the tumor-derived CCL2 via a neutralizing mAb reduced metastasis to bone and lung.151Adverse side effects of anti-CCL2 therapy have raised serious concern as it has shown to aggravate metastasis via increasing macrophage recruitment within weeks of treatment termination.