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

Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors

It is known that cancer cells have aberrant cell cycle machinery that aids them to divide and proliferate infinitely. Thus somehow, inhibiting the cell cycle in these cancer cells might provide a way to overcome the resistance in cancer cells. The proteins involved in regulating cell cycle belong to cyclin-dependent kinase (CDK) family. In addition, CDK4/6 has been shown to regulate the cell cycle progression by its reversible interaction with cyclin D1. Thus, among the emerging therapies against CDKs, CDK4/6 inhibitors such as abemaciclib, ribociclib, and palbociclib are the most promising candidates. These CDK4/6 inhibitors block the phosphorylation of retinoblastoma protein, resulting in the downregulation of E2F-response genes to mediate cell cycle arrest at the G1-S stage. These small molecule inhibitors have also been reported to dephosphorylate the forkhead box protein M1 (transcription factor), causing inhibition in cellular proliferation.¹⁹ Interestingly, it was observed that hormone-resistant tumors are still dependent on CDK4/6-cyclin D1 for their growth and proliferation.²⁰

Promising results have led to the FDA approval of combination treatment using ribociclib and palbociclib along with aromatase inhibitor as the first-line treatment for ER+/PR+/HER2− advanced BC. They have been reported to significantly improve the PFS in advanced BC patients by 10 months and the PFS rate by 20% after 18 months, respectively, compared to letrozole alone.²¹ Abemaciclib has been shown to prolong the median PFS by 7 months,²² when employed as second-line treatment in combination with fulvestrant in ER+/PR+/HER2− advanced BC. To assess the efficacy of ribociclib and abemaciclib alone, they are currently in Phase III trials (NCT02422615 and NCT02246621). Although the mechanism of action of these CDK4/6 inhibitors is alike, abemaciclib showed greater monotherapy response and induced lesser neutropenia as compared to other inhibitors, primarily due to its more specific CDK4 inhibition.²¹ In addition to the conventional/direct approach of targeting the signaling cascade involved in mediating resistance, there has been development of inhibitors which have the potential to reverse the resistance.

Histone deacetylase (HDAC) inhibitors

Resistance to conventional hormonal therapy has also been attributed to the histone deacetylation-mediated loss of ER expression in ER+ patients.¹ In line with this observation, application of HDAC inhibitors has been shown to upregulate the expression of ERα and aromatase, thereby aiding in suppression of the signaling governed by ER.²³ Entinostat when combined with exemestane and vorinostat in combination with tamoxifen has shown promising antitumor activity when employed as second-line treatment for ER+/PR+ advanced BC in combination with, compared to, their monotherapy counterparts.²³

Steroid sulfatase inhibitors

Steroid sulfatase also known as arylsulfatase C is a sulfatase enzyme involved in the metabolism of steroids. Interestingly, the enzymatic activity of steroid sulfatase was reported to be substantially increased in ERα-positive BC cells.²⁴ Thus, inhibiting the enzymatic activity reduces the estrogenic steroids and suppresses tumor growth. Encouraging results were reported from the Phase II trial of the combinatorial treatment using irosustat along with conventional aromatase inhibitor.²⁵ SR16157 (dual-acting steroid sulfatase inhibitor) which is a direct inhibitor of steroid sulfatase and releases ERα modulator has also been assessed for its effects in hormone-dependent BC.²⁶

Resistant BC against HER2 targeted therapy

The growing reports of primary and acquired resistance to lapatinib or trastuzumab are alarming and severely hampering its clinical significance as HER2+ BC therapeutics. Thus, identifying the resistance mechanisms and discovery of potential therapeutic agents to tackle the resistance is the need of the hour. Various groups have identified and verified numerous therapeutic agents that might play an important role in the fight against metastatic HER2+ BC.

PI3K/Akt/mTOR inhibitors

The aberrant activation of PI3K/Akt/mTOR pathway is also considered to be mediator of resistance in HER2+ BC, thus combining the inhibitors of this pathway with HER2 targeted candidates and studying the efficacy of the drugs is an area of active research. Buparlisib and pilaralisib (pan-class I PI3K inhibitors), when administered with lapatinib,²⁷ trastuzumab,²⁸ or trastuzumab and paclitaxel,²⁹ was proven to be efficacious and safe in patients having HER2+ advanced disease. In addition, MK-2206 (an Akt inhibitor) also showed promising antitumor activity when combined with trastuzumab and paclitaxel²⁸ or trastuzumab³⁰ alone in patients with HER2+ advanced BC. To directly target the mTOR, everolimus was combined with trastuzumab and vinorelbine; however, the clinical outcome of advanced HER2+ BC patients did not improve.³¹ Surprisingly, this combination demonstrated better anticancer activity than trastuzumab alone in HER2+ patients who are hormone receptor negative.³¹Recent drugs such as sirolimus³² and ridaforolimus³³ when administered in combination with trastuzumab have demonstrated promising results in refractory HER2+ BC.

Inhibitors targeting HER-family receptors

Receptor ligands switching between HER-family members (HER1 [EGFR], HER3, or HER4) can activate the signaling cascade, this phenomenon is reported to make trastuzumab redundant.³⁴ In addition, HER2/HER3 heterodimers have also been associated with trastuzumab resistance.³⁵ Thus suppression of the HER-family members may be promising to deal with the conundrum of resistance in HER2+ advanced BC. In view of this, an irreversible TKI, neratinib was developed that has the ability to inhibit HER1/HER2/HER4. It has been reported that administration of neratinib after trastuzumab adjuvant therapy has significantly improved the 2-year invasive disease-free survival in HER2+ patients.³⁶ Preclinical study has also shown encouraging antitumor activity of an anti-HER3 mAb (patritumab) by inhibiting HER2/HER3 heterodimers. Further, it was shown that it is efficacious and has lower toxicity in patients with advanced HER2+ disease.³⁷

A novel mAb margetuximab (HER2 targeting) was also assessed in a first Phase I trial for its antitumor activity and was found to be well tolerated and had promising activity even as a single therapeutic agent.³⁸ Further, this antibody inhibitor is currently undergoing trials to test its efficacy as a single agent (NCT02492711) and/or in combination with pembrolizumab (targets PD-1 receptor of lymphocytes) (NCT02689284).³⁸ Among other ongoing efforts, trastuzumab is conjugated with emtansine (microtubule inhibitor) which utilizes the specificity of trastuzumab for targeting HER2+ BC cells and microtubule cytotoxicity for killing the cells.³⁹ It has been approved as a second-line treatment for lapatinib/trastuzumab-relapsed/refractory HER2+ BC patients.⁴⁰

Immunotherapy

The use of immune system against tumor cells serves as an area of extensive research with the aim to develop a vaccine against cancer. One of the first devised immunotherapeutic agents was nelipepimut-S, derived from the extracellular region of HER2. It has been extensively analyzed as a potential vaccine to prevent relapse in high-risk BC patients.⁴¹ The combinatorial application of nelipepimut-S and trastuzumab in HER2+ early BC is studied in Phase IIb clinical trial (NCT02297698). Recombinant HER2 protein (dHER2) was also studied for the potential vaccine and exhibited immunogenicity to augment T-cell-mediated response against HER2+ BC.⁴² Follow-up studies are being carried out to elucidate its role as monotherapy in HER2+ advanced BC as well as advanced BC refractory to trastuzumab or lapatinib.⁴³

TNBC

Among the BC subtypes, TNBC has fewer choice of therapeutic drugs, primarily due to the lack of well-characterized molecular targets. Therefore, the need of the hour is to identify novel targets and develop effective agents against these targets to achieve improved clinical benefits. Till now the agents developed against TNBCs are primarily based on drug repurposing.

Anti-angiogenic agents

Vascular endothelial growth factor (VEGF), which is a key angiogenic factor implicated in various cancers, has been reported to be higher in TNBC as compared to non-TNBC BC.⁴⁴ A well-known anti-VEGF mAb, bevacizumab, demonstrates suppression of tumor neovasculature growth and inhibits metastasis. It was also reported in a Phase III trial that supplementation of bevacizumab to docetaxel (first-line chemotherapy) resulted in improved response rate.⁴⁵ It was also observed that the combination of bevacizumab and docetaxel had minimal or no side effects when compared to docetaxel alone.

Poly(ADP-ribose) polymerase (PARP) inhibitors

A major breakthrough toward the understanding of the heterogeneity of the TNBCs came in the form of detection of a subclass of sporadic TNBC that has deficiency in the homologous-repair pathway, which is a characteristic of BRCA1/2-mutated BC. In line with this observation, the therapeutic drugs administered to these patients incorporate PARP inhibitors or platinum drugs (DNA targeting) like carboplatin⁴⁶ along with conventional chemotherapy.⁴⁷ BRCA1/2 genes are responsible for encoding tumor-suppressor genes that are involved in repairing DNA double-stranded breaks via homologous recombination. Whereas, PARP enzymes repair the single-stranded breaks. Patients harboring germline BRCA1/BRCA2 mutation (gBRCA+) benefit the most after administration of PARP inhibitors, probably due to synthetic lethality.⁴⁸

When considering PARP inhibitors, olaparib seems to be a success story. In addition to olaparib, other inhibitors targeting PARP such as talazoparib is currently in Phase III trial (NCT01945775). Talazoparib has shown promising preclinical results, which can be attributed to its strong affinity to DNA by trapping PARP–DNA complexes.⁴⁹ It has also demonstrated strong anticancer activity as a monotherapeutic drug in advanced gBRCA+ BC.⁵⁰ Rucaparib (Phase II, NCT02505048) and niraparib (Phase III, NCT01905592) are being explored in case of gBRCA+ advanced BC patients as a single agent as well as in combination with standard chemotherapy (niraparib: Phase I/II, NCT02657889; rucaparib: Phase II, NCT01074970).

The decision of using either PARP inhibitors or carboplatin in TNBC is generally determined by three DNA-based homologous recombination deficiency scores, which highly correlates with the germline genetic defects in BRCA1/2.⁵¹ However, none of the abovementioned therapeutic agents is beneficial against all TNBC because of the heterogeneous nature of TNBC. Thereby, an urgent need for the identification and characterization of novel clinically important molecular biomarkers for further refinement of the in-practice treatment approaches.