The PI3K/AKT/mammalian target of rapamycin (mTOR) pathway has been demonstrated to be involved in secondary endocrine resistance in ER+ BC,53 and thus inhibition of this pathway is a promising approach to overcome resistance. Therapeutic blocking of this signaling pathway includes the use of PI3K inhibitors (PI3Ki) such as pan-PI3K inhibitors, which include buparlisib and pictilisib54 (Figure 1). Buparlisib is an orally available pan-PI3Ki and the most clinically advanced agent in this class. The efficacy of this PI3Ki has been confirmed in several clinical trials (NCT01339442, NCT01610284), in which the combination of buparlisib with FUL in ER+ BC patients increased prolonged progression-free survival compared with FUL alone.55 Pictilisib is an oral pan-PI3Ki of multiple PI3K isoforms. The combination of pictilisib and FUL in patients with postmenopausal metastatic BC previously treated with an AI was evaluated in a randomized Phase II clinical trial (FERGI, NCT01437566). The trial results showed no significant difference in progression-free survival with FUL + pictilisib compared with FUL + placebo. According to the authors, such results could be due to the higher toxicity from the combination treatment, including rash, diarrhea, transaminitis, and fatigue, which limits the administered dose of pictilisib.56 Based on the above results, the development of pictilisib in this setting was discontinued. Additionally, the mTOR inhibitor everolimus (Afinitor) (Figure 1) overcomes resistance to hormone therapy by controlling the AKT/mTOR signaling pathway.57 Furthermore, recent studies showed the benefit of everolimus with CDK4/6 inhibitor treatment in ER+ metastatic BC patients.58
Drug reuse or repositioning refers to the process of seeking new medical treatments within available medications, rather than developing new medications.59 This strategy reduces the approval time for drug use and increases the success of clinical development. One example of a drug that has been repurposed for BC treatment is raloxifene, which has been used for the treatment and prevention of postmenopausal osteoporosis. Raloxifene was approved by the FDA in 2010 for the primary chemoprevention of BC,60 and one study showed that raloxifene was as effective as TAM in reducing the risk of BC.61 In fact, raloxifene was associated with a reduced risk of invasive BC in postmenopausal women.62 Other repurposed drugs with possible anticancer activity in BC are listed in Table 1.
In addition, anticancer activity have also been evidenced with the use of metformin, which is used for the treatment of type II diabetes; aspirin, an anti-coagulant;63 clopidogrel, which is used for post-myocardial infarction;64 and fingolimod, a drug for multiple sclerosis.65 Although repurposed drugs have shown more benefits than conventional treatments, some professionals oppose the use of repurposed drugs.66 However, numerous studies have demonstrated the efficiency and effectiveness of repurposed drugs in reducing resistance and toxicity in patients, especially drug combinations of reused drugs and conventional drugs for BC.
Resistance and Overcoming Resistance in HER2+ BC
Drugs approved for the treatment of HER2+ BC include humanized antibodies, PI3K inhibitors, antibody-drug conjugates (ADCs), and TKIs. Additionally, several drugs for the treatment of HER2+ BC, including ADCs, are currently being reviewed by the FDA for possible approval.
Humanized Monoclonal Antibodies (mAbs)
Currently available humanized antibodies for the treatment of HER2+ BC include trastuzumab, pertuzumab, and 19H6-Hu.
Trastuzumab specifically binds to the extracellular domain of HER2 to prevent the antibody-dependent cellular cytotoxicity,67–69 inhibiting the downstream signaling of HER270 and negatively regulating HER249,71,72 (Figure 1).The introduction of trastuzumab to therapeutic regimens for patients with HER2+ BC has improved both the response and clinical outcome of these patients. However, not all patients with HER2+ BC respond to treatment and some develop resistance. The mechanisms related to resistance to trastuzumab therapy include the truncated form of HER2 (P95HER2),73 masking by epitopes,74,75 activation of the PI3K/AKT/mTOR signaling pathway,76 and FCGRIIa polymorphisms,77 among others.
In some BC patients, the HER2 protein (185 kDa) gradually loses its extracellular domain through proteolytic detachment and the remaining 95 kDa fragment (p95HER2) associated with the membrane acquires constitutive activity.78 The p95HER2 truncated form of HER2 lacks the extracellular domain, which is the binding site for trastuzumab; however, because the intracellular domain is intact (which shows strong kinase activity), some studies have associated p150 HER2 with clinical resistance to trastuzumab.73 Compared with tumors expressing the full-length HER2, p95HER2-expressing tumors have worse prognosis and are at an increased risk of metastasis.73
A mechanism of obstruction of the connection point between trastuzumab and HER2 corresponds to the masking of epitopes, with proteins Mucin 4 (MUC 4) and hyaluronic complex polymer (CD44) being involved in this mechanism.74 When MUC4 and CD44 are active, they alter the HER2 binding point,74,75 leading to a decrease in trastuzumab binding by 20%.75
The PI3K signaling pathway plays a crucial role in growth and cell survival. Somatic missense mutations in the PIK3CA gene are common in patients with HER2+ BC.76 In addition to contributing to neoplastic transformation,26 these mutations lead to alterations in the PI3K/AKT/mTOR pathway and reduced efficacy of trastuzumab therapy. Therefore, additional diagnostic testing for planning customized treatment, such as detecting PIK3CA gene mutations, have recently been approved and are used to establish personalized therapies.79
Immune effector cells, such as natural killer cells or macrophages, can recognize and bind trastuzumab through their receptors. Some of the receptors, called Fc-gamma receptors, including FCGRIIa, seem to be crucial for the clinical response to trastuzumab. Thus, the presence of polymorphisms in these receptors can modulate the response to trastuzumab and result in the development of resistance.77
Pertuzumab is a second-generation recombinant humanized monoclonal antibody that binds to the extracellular dimerization domain II of HER2, preventing its heterodimerization with HER1, HER3, HER4 and IGF-1R31,80 and thus inhibiting cell proliferation (Figure 1). Pertuzumab has been associated with both increased progression-free survival for patients with metastatic BC81 and better outcomes for patients with early BC. However, in patients with non-visceral metastases or small primary tumors with negative nodes, the therapeutic benefit of pertuzumab is relatively small.82
Based on the low response rates of BC patients to trastuzumab therapy, new treatment strategies were explored. The new anti-HER2 antibody (19H6-Hu), which enhances the anti-tumor efficacy of trastuzumab and pertuzumab with a distinct mechanism of action, has shown promising efficacy. 19H6-Hu is a novel humanized anti-HER2 monoclonal antibody that binds to the HER2 extracellular domain with high affinity (Figure 1) and inhibited the proliferation of multiple HER2+ BC cell lines as a single agent or in combination with trastuzumab. One study showed that 19H6-Hu in combination with trastuzumab was more effective at blocking phosphorylation of ERK1/2, AKT (S473) and HER2 (Y1248) in HER2+ BC cells compared with trastuzumab alone or in combination with pertuzumab.83
PI3K inhibitors are used in combination with AIs for the treatment of metastatic BC.84 However, most PI3K inhibitors are more toxic than beneficial. The FDA approved more selective, less toxic, and more effective inhibitors for the treatment of metastatic BC in patients with PIK3CA gene mutation, such as alpelisib and taselisib85 (Figure 1). Additionally, another study reported that PI3K/AKT/mTOR inhibitors in combination with trastuzumab or trastuzumab and paclitaxel were efficient and safe for patients and showed beneficial anti-tumor activities.86
ADCs are a means of delivering cytotoxic drugs specifically to cancer cells. The mechanism of action of ADCs involves the delivery and subsequent internalization of the ADC and the release of highly active, free cytotoxic agents into cancer cells, ultimately leading to cell death. ADCs used in the treatment of advanced BC include trastuzumab emtansine (T-DM1)87 and trastuzumab deruxtecan88 (Figure 1). T-DM1 is a conjugate of trastuzumab and a cytotoxic drug (DM1, derived from maytansine) that is effective and generally well tolerated when administered as a single agent. The efficacy of this ADC has been demonstrated in randomized trials.89 Although the superior efficacy of T-DM1 compared with trastuzumab or trastuzumab plus chemotherapy has been reported in the treatment of metastatic BC, most patients treated with T-DM1 eventually show disease progression,87 and some HER2+ BCs do not respond or only respond minimally to T-DM1.87 One study showed that altered traffic/metabolism of T-DM1 is one of the predominant mechanisms associated with resistance to T-DM1, and a greater understanding of such mechanisms is necessary to develop strategies to overcome T-DM1 resistance.90
Another ADC used in the treatment of BC is trastuzumab deruxtecan. This conjugate comprises an anti-HER2 antibody, a cleavable tetrapeptide-based linker, and a cytotoxic topoisomerase I inhibitor. Although the use of this ADC in a pretreated patient population with HER2+ metastatic BC showed durable antitumor activity, the efficacy of this ADC in HER2+ metastatic BC patients previously treated with TDM-1 needs to be confirmed.88 Trastuzumab deruxtecan is also being evaluated for the treatment of metastatic BC in patients with low HER2 expression, for whom current available therapies targeting HER2 are ineffective. The results of trastuzumab deruxtecan treatment in these patients have shown a response rate of 44.2%.91
Currently, several HER2-directed ADCs are under clinical investigation for both HER2 amplified and HER2 expressing but not amplified BCs, including ARX788 and XMT-1522.ARX788, a novel next-generation anti-HER2 ADC containing an anti-HER2 monoclonal antibody site-specifically conjugated to amberstatin,92,93 has shown anti-tumor effects and rapid tumor regression in murine xenograft models of the HER2+ BC cell lines BT474 and HCC1954.92 Furthermore, ARX788 showed a stronger inhibitory effect than T-DM1 on T-DM1-responsive BC cells and caused complete tumor regression in a trastuzumab-resistant BC xenograft model derived from JIMT-1 cells.94 Two Phase 1 trials on of ARX-788 (clinicaltrials.gov identifiers: NCT02512237 and NCT03255070) are ongoing but the results have not yet been published.95 XMT-1522 is a ADC containing a human IgG1 anti-HER2 monoclonal antibody (HT-19) that binds to domain IV of HER2 to an epitope that is distinct from the trastuzumab binding site.96 Notably, the HT-19 antibody does not compete with trastuzumab or pertuzumab for binding to HER2.97 Clinical studies on a panel of 25 tumor cell lines differentially expressing HER2 showed that XMT-1522 was more potent than T-DM1 in inhibiting cell proliferation.97 Additionally, in a HER2 BC BT474 xenograft model and a patient-derived HER2 xenograft model, treatment with XMT-1522 induced complete tumor regression at doses of 2 mg/kg and 1 mg/kg, respectively.97 Benefits of XMT-1522 therapy were also observed in a BC xenograft model, in which a significant inhibition of tumor growth was observed.98 Antitumor efficacy, with complete response, was also observed in some mice after the combined treatment of XMT-1522 with the anti-PD1 monoclonal antibody; the response was better when the two drugs were administered sequentially (XMT-1522 followed by anti-PD1 monoclonal antibody).95 The use of XMT-1522 is currently being investigated in patients with advanced BC expressing HER2 progressing on standard therapy (clinicaltrials.gov identifier: NCT02952729).
Other ADCs currently under preclinical and clinical research for the treatment of BC include A166, ALT-P7, DHES0815A, DS-8201a, RC48, SYD985, and MEDI4276. However, to the best of our knowledge, there are no published data available for A166, ALT-P7, and DHES0815A at the time of writing.
TKIs used to treat HER2+ BC include lapatinib, neratinib, and tucatinib (Figure 1). Lapatinib, a potent and reversible small molecule TKI that binds both HER2 and EGFR, inhibits the growth of trastuzumab-resistant HER2+ tumor cells.99 Neratinib, an irreversible small-molecule TKI of HER1, HER2 and HER4,100 has been shown to be effective as a single agent in the treatment of HER2+ metastatic BC pretreated with trastuzumab.101
Tucatinib is an investigational, oral TKI that is highly selective for the kinase domain of HER2 with minimal inhibition of EGFR.102 Combinations between tucatinib and monoclonal antibodies (such as trastuzumab) have been recently developed for the treatment of HER2+ metastatic BC patients. For instance, a non-randomized open-label phase 1b study found that the combination of tucatinib with trastuzumab and capecitabine showed encouraging antitumor activity in patients with HER2+ BC, including those with brain metastases. However, side effects such as diarrhea, nausea, palmo-plantar erythrodysesthesia syndrome, fatigue, and vomiting were observed.103 Additionally, in heavily pretreated patients with HER2+ metastatic BC, the combination of tucatinib plus trastuzumab and capecitabine resulted in better progression-free survival and overall survival outcomes.104
Resistance and Overcoming Resistance in TNBC
Treatment of TNBC mainly involves the use of chemotherapy. Additionally, given the immunogenicity of TNBC, this type of cancer can respond to immunotherapy. New therapeutic options are currently being developed for this disease, including ECT, androgen antagonists such as bicalutamide and abiraterone acetate,105 and nanosomal docetaxel lipid suspension (NDLS)–based chemotherapy.106
Despite the associated short- and long-term risks, chemotherapy remains essential to prevent recurrence in many patients with advanced BC. Chemotherapy is the only systemic therapy with proven efficacy in TNBC and an important complement to endocrine therapy or HER2-targeted therapy in patients with hormone receptor-positive (ER+ and PR+) BC.2 In fact, the first-line implementation of taxanes has been associated with optimization of the immune status of BC patients and therefore with a good clinical response. Optimization of immune status has been associated with increased activity of natural killer and lymphokine-activated killer cells, with increased levels of interleukin-6 (IL-6) and reduced levels of IL-1 and tumor necrosis factor.107 Chemotherapeutics include the use of alkylating agents, antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors, TKIs, and mitotic inhibitors.108
Resistance to chemotherapy is a result of chronic exposure to chemotherapy and increases the chances of recurrence and accelerated metastasis.109 Therefore, resistance is a major limitation for the successful treatment of BC. However, the exact mechanisms leading to such resistance remain unclear.110 Several mechanisms associated with the resistance to chemotherapy are described below.
Membrane Glycoproteins That Act as Effusion Pumps
Membrane glycoproteins that act as effusion pumps are ATP hydrolysis–dependent proteins that actively transport different substrates across the cell membrane. The ATP-dependent transporters or ABC transporters (ATP-binding cassette) include multidrug resistance proteins (MRP) such as the permeability glycoprotein (P-GP). These transport proteins are involved in the most widely recognized drug resistance mechanism.111 However, recent studies show that FUL reverses resistance to doxorubicin (DOX) mediated by glycoprotein transporters in hormone receptor-negative BC cells. The reversal of resistance occurs through the sensitization of tumor cells potentiating DOX cytotoxicity by increased intracellular drug, and further activation of apoptosis and cell cycle arrest34 (Figure 3A).
The amount and affinity of receptors present in the membrane determine the effectiveness of the drug, allowing it to be paired or not (Figure 3B).
Enzyme System That Deactivates Anticancer Drugs
The enzyme system involved in the deactivation of anti-cancer drugs, and therefore involved in the process of drug resistance,111,112 includes multi-drug resistance gene (MDR1) and glutathione S transferase Pi (GSTP1). While GSTP1 decreases the concentration and the effective life of drugs, resulting in drug inefficiency,113 MDR1 gene is significantly overexpressed in multidrug resistance phenotype114 (Figure 3C).
Epigenetic mechanisms, such as DNA methylation and histone modifications, play a key role in the regulation of gene expression (silencing of tumor suppressor genes and/or overexpression of oncogenes)110,115 (Figure 3D). Alterations in epigenetic mechanism have not only been associated with the acquisition of resistance but are also involved in tumor progression and metastasis.109 The application of epigenetic therapies, such as hydralazine and valproic acid, has been reported to achieve a significant reversal of acquired resistance to chemotherapy.110 In fact, it has been indicated that the molecular effects exerted by valproic acid and hydralazine include the inhibition of histone deacetylases, the demethylation of DNA and the reactivation of genes in primary tumors of patients with BC, increasing the efficacy of chemotherapy.116
The tumor microenvironment includes stromal cells (fibroblasts, vascular cells, and immune system cells), soluble factors (such as growth factors, transcription factors, hormones, and cytokines), extracellular matrix, signaling molecules, hypoxia (which facilitates the release of exosomes), and mechanical signals (exosomes).109,117 In addition to creating favorable niches for metastasis, these factors facilitate the transfer miRNA, cytokines, and P-GP from resistant cells, altering the gene expression of sensitive cells, thereby increasing their survival.112,117,118 Tumors are usually exposed to hypoxic conditions; therefore, to obtain energy, they must rely on glycolysis, which turns chemotherapeutic drugs ineffective by the increased expression of metabolic enzymes. In fact, Tsuruo et al119 reported that hypoxia is a trigger for tumor resistance to chemotherapy, since it leads to both decreased DNA topoisomerase II alpha (TOP2A) expression and upregulation of MRP expression (Figure 3E).
The twist family bHLH transcription factor 1 (TWIST1) plays a key role in angiogenesis. TWIST1 positively regulates EMT,109 invasion, and metastasis.118 The TWIST1 transcription factor can recognize the E-box gene sequence on the promoters of E-cadherin and depress its transcription, thereby leading to decreased cell adhesion and promoting angiogenesis.113 In addition, was reported that up-regulation of TWIST1 by NF-κB contributes to the chemoresistance113,115 (Figure 3F).
Pharmacokinetics are associated with the reduction of intracellular drug accumulation because of increased drug efflux, causing the generation of reactive oxygen species and detoxification mediated by exosomes.110,112,120 Pharmacodynamics may confer chemoresistance because it leads to alterations of the drug target,110 repression of tumor suppressor genes, impaired DNA damage repair, acquisition of characteristics similar to cancer stem cells, cellular changes induced by EMT for angiogenesis, and tumor microenvironment conditions.113 The significant reduction in survival rates for repeated relapse patients is the most concerning issue associated with pharmacokinetics.
Resistance to Treatment with Anthracyclines: DOX
Anthracyclines, such as DOX, are one of the most widely used drugs in the treatment of metastatic BC as a single-agent therapy. Anthracyclines are DNA damaging agents and inhibit TOP2A. DOX intercalates into DNA, preventing TOP2A binding and causing replication fork blockage,117 which eventually leads to apoptosis.10 DOX is used as the first-line treatment in BC and primarily for the treatment of advanced BC with an overall response rate of 30%–50%,120 alone or in combination therapy with paclitaxel,110 docetaxel (DOC), cyclophosphamide, 5-fluorouracil, trifluridine, or vorinostat. Despite its extensive use, DOX is an antitumor drug with high cytotoxicity.110 The cardiotoxicity mechanisms of DOX remain to be explained; however, formation of DNA adducts and free radicals have been reported as potential mechanisms.120
Diverse hypotheses have been formulated around DOX resistance, including resistance related to BC tumor subtypes,121 resistance related to the altered expression of specific proteins, including NFκB and small modifier proteins like ubiquitin (SUMO), and resistance related to epigenetic modifications. High levels of IL-6, IL-8, IL-1β, transforming growth factor beta (TGF-β), and the prion protein (PrPc) have also been associated with resistance to DOX.117
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