Resistance and Overcoming Resistance in Breast Cancer

Resistance to Treatment with Taxanes: Docetaxel (DOC)

DOC is an antineoplastic agent; its mechanism of action involves inhibiting cell division by stabilizing microtubules, inhibiting tubulin depolymerization, and killing tumor cells.¹⁰⁸ DOC is one of the most active drugs available for the treatment of metastatic BC.¹²² DOC is systematically applied in patients who do not respond or develop resistance to chemotherapy with anthracyclines (such as DOX).^3,108 The risks and long-term benefits of chemotherapy are poorly understood because many studies do not report long-term results or do not monitor therapeutic resistance.¹²⁰ However, in recent prospective trials on neoadjuvant chemotherapy, taxanes along with anthracyclines have been observed to increase life expectancy by metastasis-free survival in more aggressive tumors.¹¹⁸

Resistance to DOC has been mainly associated with miRNA transport and membrane glycoproteins via exosomes, which also promotes high drug efflux from the cell interior to the exterior.¹¹² DOC resistance is highly influenced by the presence of cytokines. For example, IL-8 leads to increased NFκB and is involved in the activation of survival pathways such as those mediated by tumor necrosis factor receptor 2 (TNFR2), whereas high levels of IL-17A lead to increased cell proliferation and thus resistance.¹¹⁷

Immunotherapy

Considering that TNBC has the highest frequency of mutations and therefore the highest possibility of expressing immunogenic neoantigens, this BC subtype is considered to have the highest probability of responding to immunotherapy.¹²³ Cancer immunotherapy aims to overcome the ability of tumor cells to resist the endogenous immune response by stimulating the patient’s immune system. Immunotherapy mainly involves the use of mAbs that are selectively directed to a specific target involved in tumor cell proliferation.¹²⁴ Among the mAbs used in BC, the synthetic antigen sialyl-Tn has been proven to be safe and highly efficient because it triggers a large immune response.¹²⁵

Other mAbs include bevacizumab, an antibody that inhibits the activation of VEGF receptor, thereby increasing the rate of drug response and progression-free survival. Other mAbs, such as aflibercept (a recombinant fusion protein) and pazopanib (a TKI) have shown good results in the inhibition of BC tumor cell migration and metastasis. Some mAbs substantially improve the effects of DOX in tumor cells expressing high levels of EGFR.¹²⁶

In recent years, anti-PD-1 receptor antibodies have been considered incredibly promising in the field of cancer immunotherapy. The PD-1 receptor and its ligands PD-L1/PD-L2 belong to the family of immune control proteins and act as co-inhibitory factors to modulate the response of T cells and to prevent chronic autoimmune inflammation.^126,127 Anti-PD-1 mAbs (such as pembrolizumab and nivolumab), which are known as immune checkpoint inhibitors, block the PD-1 receptor or PD-L1 and PD-L2 ligands expressed by cancer cells and prevent their binding; this leads to an inhibition of the immune modulatory signal and allows T cells to remain active against tumors. However, a limited efficacy of these mAbs (targeting PD-1 and PD-L1) has been reported, mainly owing to the induction of apoptosis in patients with metastatic BC because of the low proportion of lymphocytes in infiltrating tumors. Therefore, a combination of immunotherapy and targeted therapies such as angiogenesis inhibitors is used for BC patients.^126,127

ECT

ECT was developed as a treatment option for TNBC patients, given the absence of viable therapeutic targets in this BC subtype. ECT involves the efficient delivery of natural bioactive molecules with anti-cancer effects via a biophysical means and is based on the combined use of electroporation together with chemotherapy drugs to increase absorption. Recent research has demonstrated the efficacy of ECT and showed that ECT can dramatically increase the intracellular concentration and cytotoxicity of several FDA-approved drugs, such as bleomycin and cisplatin, leading to a complete tumor response with minimal drug doses.¹²⁸ Additional studies in BC cell lines showed that ECT increased the cytotoxicity of curcumin treatment by 7-fold,¹²⁹ increased apoptosis in MDA-MB-231 cells, and minimally affected the viability of non-cancerous MCF10A cells.^129,130 A recent study demonstrated the anticancer effects of ECT against the MDA-MB-231 cell line, which is representative of the TNBC subtype.¹³¹ These effects include the suppression of key proteins involved in the proliferation, differentiation, migration, survival, and apoptosis of cancer cells.

Emerging Treatments

In recent years, emerging treatments are being developed that could offer additional benefits to conventional treatments that are mainly related to overcoming resistance and decreased side effects. These treatments involve nanotechnological approaches (Figure 1), CRISPR, exosomes, and miRNAs.

Nanotechnological Approaches

Nanotechnological approaches are being developed to minimize the toxicity in healthy tissues generated by targeted therapy while maintaining the efficacy of the treatments. These approaches are based on a nanoparticle delivery system (nanocarriers) that increases the drug-site contact time and the elimination time and reduces the resistance to the drug. Examples of nanocarriers in BC therapy include nanoparticles, polymeric micelles, dendrimers, carbon nanotubes, liposomes, and quantum dots^107,112,113 (Figure 1).

The NDLS-based chemotherapy system was recently developed. NDLS is a new formulation of lipid-based, polysorbate 80 and ethanol-free formulation of docetaxel developed to overcome toxicity problems.¹³² The function of NDLS is based on its ability to infiltrate and become entrapped in the damaged tumor vasculature and collagen material of necrotic tumor tissue, leading to increased drug retention (leading to an enhanced permeability and retention effect).¹⁰⁶ NDLS showed comparable efficacy and tolerability to conventional DOC in the treatment of metastatic BC in both prospective and retrospective studies.¹³² A recent study demonstrated that NDLS-based chemotherapy was effective and well tolerated in the treatment of patients with BC in all settings (neoadjuvant, adjuvant, and metastatic).¹⁰⁶ The efficacy of NDLS is currently being prospectively evaluated in patients with TNBC (ClinicalTrials.gov identifier: NCT03671044).

CRISPR

Given the wide variety of morphological and molecular features of tumor cells, the identification of new therapeutic targets to prolong survival and limit the acquisition of resistance in patients with BC is crucial. Implementation of genomics edition by CRISPR system has provided a glimpse of broad cellular heterogeneity and the opportunity to act on tumor cells with mutant alleles,^133,134 such as alterations in EGFR, KRAS, BAP1, BRAF, BRCA1, and BRCA2, or with replicative immortality without affecting normal cells.^133,135 The CRISPR system (CRISPR/Cas9) has been established in preclinical trials as an effective tool to specifically attack and/or resensitize tumor cells.¹³⁶

Potential targets have been identified in BC through CRISPR/Cas9, including microtubule affinity regulating kinase 4 (MARK4) and fermitin family homolog-2 (FERMT2), which are related to metastasis; microtubule-associated serine/threonine kinase (MASTL), which is involved in cell proliferation; the tyrosine protein kinase FYN that is involved in drug resistance; and cyclin-dependent kinase 7 (CDK7), which is negatively associated with tumor size and grade.¹³⁵ CRISPR/Cas9 has also been used to generate changes in exons of the HER2 gene. Such changes lead to the expression of a truncated HER2 protein that prevents dimerization of the transmembrane domain, leading to inhibition of signaling pathways such as MAPK/ERK and PI3K/AKT, as well as cell proliferation and tumorigenicity.¹³⁴ As with immunotherapy, CRISPR/Cas9 can also target stromal cells in the tumor microenvironment to generate mutations in VEGF and VEGFR2, thereby inhibiting stromal cell migration activity.¹³⁶

Exosomes

Exosomes, an important class of extracellular vesicles, transport and distribute biological macromolecules, such as proteins, nucleic acids, and anti-tumor drugs, and are considered promising therapeutic strategies for targeted drug delivery in BC.¹³⁷ The therapeutic potential of exosomes as nano-transporters of DOX has been considered as a strategy to overcome resistance to chemotherapy.¹³⁸ The use of exosomes could allow the targeted administration of drugs, overcoming several limitations associated with conventional nanoparticles, including cytotoxicity, drug modification, greater synergistic effects, and biocompatibility, among others.^107,112,113

miRNAs

Several miRNAs have been identified as biomarkers for prognosis and treatment planning in BC.¹³⁹ miRNAs also play a crucial role in the regulation of BC cell sensitivity to chemotherapy.^140,141 For example, miR326 and miR-451 negatively regulate the expression of MDR genes, including MDR-1, to overcome resistance. MDR-1, which encodes the P-GP transporter, multi-drug resistance proteins (MRPs), and the BC resistance protein (BCRP), which transport both hydrophilic and hydrophobic substrates, are particularly important for chemotherapy.¹¹¹ Over 80% of the currently used anticancer agents are transported by P-GP, and thus its overexpression can lead to multi-drug resistance.¹¹³ Several reports have identified miRNAs that play important functions in BC (Table 2).

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