Targeting Stem Cells in the Realm of Drug-resistant Breast Cancer

EGFR inhibitors

The EGFR has been reported to be overexpressed in TNBC. Therefore, numerous clinical trials are underway to evaluate the antitumor activity of cetuximab in combination with platinum-based drugs like cisplatin in metastatic TNBC patients.^52,53 Identification of a subpopulation of TNBC patients that might respond well to EGFR inhibitors is an area of active research efforts.⁵⁴ Low expression of α-crystalline B chain, lack of KRAS expression, and higher expression of PTEN in tumors might be correlated with favorable response.⁵⁴

SRC inhibitors

SRC is a non-receptor signaling kinase which is a downstream molecule of several growth factor receptors such as PDGFR, EGFR, HGFR, and IGF-1R which have been reported to be deregulated in TNBC. Dasatinib, when tested as monotherapy for TNBC in Phase II trial (CA180059), showed substandard result.⁵⁵ However, when tested in cell lines, dasatinib in combination with anti-EGFR mAb: cetuximab and cisplatin showed synergistic antitumor activity in different TNBC cell lines.⁵⁶ The combination of three drugs resulted in more prominent induction of apoptosis and inhibition of MAPK and EGFR phosphorylation than all the other combinations.⁵⁶ In addition, cancer cell migration and invasiveness were also substantially suppressed by dasatinib as well as combination treatment with dasatinib, cetuximab, and cisplatin in TNBC cell lines.⁵⁶ Thus, clinical investigations are required to further access the use of dasatinib-containing amalgamations in TNBC patients that have tumors expressing both EGFR and c-Src.

Monoclonal antibodies

Glembatumumab vedotin is a mAb conjugated with a cytotoxic drug aimed at targeting glycoprotein NMB-overexpressing (gpNMB+) TNBC.⁵⁷ gpNMB is a transmembrane protein that has been linked with tumor invasion and promote metastasis and is overexpressed in about 40% of TNBC.⁵⁸ Phase II trial done on gpNMB+ advanced TNBC patients showed significant improvement in PFS and OS in glembatumumab vedotin-treated patients as compared to conventional therapy.⁵⁹

Breast cancer stem cells (BCSCs): the troublemakers

BC is widely understood as a heterogeneous disease which in turn contributes to therapy failure and disease progression.⁶⁰ There is not only intratumoral heterogeneity, that is, diversity within a tumor in context to phenotypic, functional, and genetic variations, but also intertumoral diversity, that is, the diversity between primary and metastasized tumor. To explain the intratumoral heterogeneity, two theories have been put forward. The first one was clonal evolution theory/stochastic theory, introduced by Peter Nowell, according to which cancer is an evolutionary process in which most neoplasms arise from single cell and progression of tumor results from stepwise accumulation of mutations within original clones following selection of more aggressive subclones. Accordingly, each dominant subclone possess similar tumorigenic potential.⁶¹

The second theory proposed is CSC theory. According to this hypothesis, only a small population of cells, called CSCs, are capable of self-renewal and have the potential to initiate tumor. In CSC model, cancers originates from the malignant transformation of a stem or progenitor cells through the deregulation of self-renewal program or from transformation of committed cells through dedifferentiation of mature cells that gain a self-renewal potential.⁶²

The first CSCs from solid tumors were identified in breast tumors,⁶³ subsequently CSCs were isolated from other organs. Al-Hajj et al were the first to identify a subpopulation of BC which had the potential to form tumors in immune-deficient Nonobese Diabetic (NOD)/Severe Combined Immunodeficiency (SCID) mice.⁶³ They used a set of cell surface markers to isolate cells with increased tumorigenic capacity. In particular, cells that were CD44+CD24^lowEpCAM+ and lineage negative (cells lacking markers CD2, CD3, CD10, CD16, CD18, CD31, CD64, and CD140b), isolated from one primary breast tumor and eight metastases, were able to form heterogeneous tumors eight out of nine times and were termed as BCSCs. Surprisingly, as few as 200 CD44+CD24^lowEpCAM+lin- cells transplanted into NOD/SCID mice could form tumors with 100% efficiency, while CD44⁻CD24+EpCAM⁻ cells could not form tumors. Different subtypes of BC constitute different proportion of BCSCs contributing to different disease outcome. Among the BC subtypes, the highest amount of CSCs was observed in patients with TNBC (basal) subtype and has been correlated with its aggressiveness.⁶⁴

With momentous discovery of CSCs, their pivotal role in driving key processes during cancer development such as tumor growth, metastasis, recurrence, as well as treatment resistance was established. However, the signaling pathway that regulates CSCs and that might be involved in promoting the resistance toward the conventional therapies remains largely elusive. Hedgehog, Notch, and Wnt pathways have been shown to play crucial role in promoting resistance to therapy. These pathways are generally involved in the development of embryo and adult tissue homeostasis. Deregulation of the Notch and Hedgehog pathways, which normally regulates stem cell self-renewal and differentiation, results in BCSC phenotype.⁶⁵ The Wnt pathway plays an important role in maintaining and preserving undifferentiated state of stem cells.⁶⁶ Hedgehog pathway, which is an embryonic development organizer pathway, is also deregulated in BC, thereby activating Gli1 and Ptch1 genes (positive modulators of the hedgehog pathway) and thus leading to BCSC proliferation.⁶⁷The Notch pathway is involved in cell differentiation during both embryogenesis and adulthood. Notch pathway deregulation activates genes important for regulating proliferation and apoptosis inhibition in cancer cells.⁶⁸ The transcription factors targeted by Notch signaling include CDKN1A, cyclinD1, c-myc, and HES-related repressor protein. These pathways have been reported to be activated in BCSCs.⁶⁹ In addition, other transcriptional factors involved in maintaining the potency of BCSCs have also been identified. The transcriptional factors such as Sox2, Oct4, and Nanog act as master regulators of pluripotency and maintain the undifferentiated state of BC cells.⁷⁰ Of the basal-like breast carcinomas, 43% exhibit higher Sox2 expression, indicating a less differentiated phenotype.⁷¹ Another member of the Sox family, Sox4, induces changes associated with the EMT process that is responsible for increased invasiveness and mobility of cancer cells in vivo.⁷² Recently, the ability of BCSCs to undergo EMT has been scrutinized, leading to the identification of partial EMT. Reports suggest that the circulating tumor cells (CTCs) survive in blood by exhibiting both epithelial and mesenchymal (E/M) phenotypes. The CTCs employ the collective cell migration properties of the epithelial cells and enhance their attachment to the extracellular matrix by achieving mesenchymal properties.⁷³ This significantly enhances the chance of survival and promote distant metastasis. Several evidences suggest that the expressions of Oct3/4, Nanog, and Sox2 are strongly associated with different CSCs, including BCSCs.⁷⁴

Apart from the genes that maintain the potency of stem cells, the BCSCs can be distinguished based on the following unique features:

1. Presence of classical cell surface marker such as CD44+CD24⁻, in addition CD133, CD44+ CD49 f^hi CD133/2^hi. CD49f and CD61 have also been introduced as BCSC marker.⁷⁵ These markers can be detected by flow cytometer, via employing specific mAbs.

2. High expression of BC resistance protein 1, also known as ATP-binding cassette (ABC) transporter G family ABCG2 or CD338.⁷⁵ This can be tested by using orthodox side population assay.

3. Ability to form mammospheres in suspension culture and the overexpression of aldehyde dehydrogenase-1 (ALDH1).⁷⁵

Chemoresistance to the conventional therapies can be divided into two main groups, namely intrinsic resistance due to genetic alterations and extrinsic resistance including microenvironment influences (Figure 3).⁷⁶ Intrinsic resistance includes overexpression of ABC transporter, overexpression of ALDH1, enhanced DNA repair mechanism, an altered cell cycle, and resistance to apoptosis. The extrinsic cause of resistance includes all microenvironment influences such as hypoxia or EMT.

Intrinsic factors of resistance

The small pool of cells, that is, BCSCs that evade chemotherapy is possibly because of the presence of ABC transporters. Increased level of ABCG2 in BCSCs was shown to enable rapid expulsion of cytotoxic drugs, conferring cellular resistance to antitumor drugs.⁷⁷ Increased levels of P-glycoprotein which belongs to ABC transport family also confer resistance to antineoplastic drugs by manipulating several cellular processes like the p53 network which plays a role in mediating chemoresistance. New tumors arising from BCSCs show a chemoresistant phenotype and are often accompanied by activating mutations.⁷⁵ Hu et al⁷⁸ observed that Akt signaling altered the subcellular localization of BCRP, thereby regulating drug efflux activity in CSCs. Inhibitors of PI3K, blocked Akt signaling, resulted in the suppression of cancer cell proliferation, but also enhanced the sensitivity of chemoresistant cells.⁷⁸

Aldefluor assays indicated that highly tumorigenic BC cells were ALDH positive. These BCSCs had the similar differentiation and self-renewal properties when compared to CSC.⁷⁹ ALDH1A1 and ALDH3A1 are important in the protection and the differentiation of CSCs via the conversion of retinol to retinoic acid.⁸⁰ ALDH1 has the ability of metabolizing toxic chemotherapeutic agents into nontoxic molecules, particularly cyclophosphamide class, by converting aldophosphamide to carboxyphosphamide and thus eliminating the lethal effects of the acrolein and phosphoramide mustard (metabolite of cyclophosphamide).^80,81 It has been observed that metastatic breast tumors overexpress ALDH.⁸²

Alteration of cell cycle kinetics is another alternative intrinsic mechanism of resistance reported in BCSC.^83,84 This feature aids the BCSC to evade death due to chemotherapeutic agents targeting rapidly dividing cells.^76,81 This quiescent state of BCSC is also responsible for relapsed disease after a long-periods of time. A dexterous DNA repair mechanism in the BCSCs is another example of intrinsic resistance mechanism.⁸⁴ BCSC uses the augmented activity of ChK1 and ChK2 allowing them to escape from mitotic catastrophe and to repair their damaged DNA proficiently.^84,85 This state of dormancy and robust DNA repair mechanism contributes to the resistance of BCSC against standard chemotherapeutic regimes.

In recent times, miRNAs are also shown to govern and regulate the BC resistance against the standard therapies. miRNAs are short, non-coding RNAs that regulate crucial biological processes and are frequently deregulated in cancer. Suppression of miR200c has been shown to promote tumorigenicity of BCSCs and normal mammary stem cells. In addition, it was shown that suppression of miR-200c triggers migration and invasion of cancer cells in the neighboring tissues.⁸⁶ Loss of miR-205 in BCSC populations has been shown to result in drug resistance properties.⁸⁷ Another report demonstrated that miR-141 is inhibited in BCSCs, contributing to the dedifferentiation of BC cells into stem-like cells which in turn enhances the stem population.⁸⁸ Alternatively, reduced expression of miR-34a in human BC resulted in inhibition of stem cell properties. A report has shown that miR-34a regulates Notch-1 pathway in sustaining stem cell properties of BCSC populations, thereby suggesting that the miR-34a/Notch-1 pathway might be a potential therapeutic target for treating BC.⁸⁹ Further, enhanced expression of let-7 miRNA has been shown to be involved in tumorigenesis of BCSCs. Other report has revealed that isoform let-7c along with Wnt signaling cascade regulates BCSC renewal in vivo.⁹⁰ In addition, miR-1 has been shown to be associated with Wnt signaling pathway which is critical for the aggressiveness of BC.⁹¹ Most of the knowledge gained in recent times has provided a foundation for the future development of miRNA-based therapy against BCSCs.