Three-dimensional culture models to study drug resistance in breast cancer

Despite recent advances in breast cancer treatment, drug resistance frequently presents as a challenge, contributing to a higher risk of relapse and decreased overall survival rate. It is now generally recognized that the extracellular matrix and cellular heterogeneity of the tumor microenvironment influences the cancer cells' ultimate fate. Therefore, strategies employed to examine mechanisms of drug resistance must take microenvironmental influences, as well as genetic mutations, into account. This review discusses three-dimensional (3D) in vitro model systems which incorporate microenvironmental influences to study mechanisms of drug resistance in breast cancer. These bioengineered models include spheroid-based models, biomaterial-based models such as polymeric scaffolds and hydrogels, and microfluidic chip-based models. The advantages of these model systems over traditionally studied two-dimensional tissue culture polystyrene are examined. Additionally, the applicability of such 3D models for studying the impact of tumor microenvironment signals on drug response and/or resistance is discussed. Finally, the potential of such models for use in the development of strategies to combat drug resistance and determine the most promising treatment regimen is explored.     

Analysis of different HER‐2 mutations in breast cancer progression and drug resistance

Studies over the last two decades have identified that amplified human epidermal growth factor receptor (HER‐2; c‐erbB‐2, neu) and its overexpression have been frequently implicated in the carcinogenesis and prognosis in a variety of solid tumours, especially breast cancer. Lots of painstaking efforts were invested on the HER‐2 targeted agents, and significantly improved outcome and prolonged the survival of patients. However, some patients classified as ‘HER‐2‐positive’ would be still resistant to the anti‐HER‐2 therapy. Various mechanisms of drug resistance have been illustrated and the alteration of HER‐2 was considered as a crucial mechanism. However, systematic researches in regard to the HER‐2 mutations and variants are still inadequate. Notably, the alterations of HER‐2 play an important role in drug resistance, but also have a potential association with the cancer risk. In this review, we summarize the possible mutations and focus on HER‐2 variants’ role in breast cancer tumourigenesis. Additionally, the alteration of HER‐2, as a potential mechanism of resistance to trastuzumab, is discussed here. We hope that HER‐2 related activating mutations could potentially offer more therapeutic opportunities to a broader range of patients than previously classified as HER‐2 overexpressed.     

Overcoming trastuzumab resistance in HER2-positive breast cancer using combination therapy

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) comprises around 20–30% of all BC subtypes and is correlated with poor prognosis. For many years, trastuzumab, a monoclonal antibody, has been used to inhibit the HER2 activity. Though, the main resistance to trastuzumab has challenged the use of this drug in the management of HER2-positive BC. Therefore, the determination of resistance mechanisms and the incorporation of new agents may lead to the development of a better blockade of the HER family receptor signaling. During the last few years, some therapeutic drugs have been developed for treating patients with trastuzumab-resistant HER2-positive BC that have more effective influences in the management of this condition. In this regard, the present study aimed at reviewing the mechanisms of trastuzumab resistance and the innovative therapies that have been investigated in trastuzumab-resistant HER2-positive BC subjects.     

LncRNA POU3F3 promotes proliferation and inhibits apoptosis of cancer cells in triple-negative breast cancer by inactivating caspase 9

It has been reported that lncRNA POU3F3 was upregulated in esophageal squamous-cell carcinomas, indicating its role as an oncogene in this disease. However, the mechanism of its function and its involvement in other malignancies is unknown. In the present study we found that expression levels of lncRNA POU3F3 were higher in tumor tissues than in adjacent healthy tissues of triple negative breast cancer (TNBC) patients and were significantly and inversely correlated with levels of cleaved caspase 9 only in tumor tissues. In addition, plasma levels of lncRNA POU3F3 were higher in TNBC patients than in healthy controls and were significantly and inversely correlated with levels of cleaved caspase 9 only in TNBC patients. In addition, treatment of exogenous Cleaved Caspase-9 significantly attenuated the effects of lncRNA POU3F3 overexpression on cancer cell proliferation and apoptosis. lncRNA POU3F3 may promote proliferation and inhibit apoptosis of cancer cells in triple-negative breast cancer.     

Wnt pathway targeting reduces triple-negative breast cancer aggressiveness through miRNA regulation in vitro and in vivo

Triple-negative breast cancer, devoid of estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER-2) expression, is deprived of commonly used targeted therapies. MicroRNAs (miRNAs) are undergoing a revolution in terms of potentially diagnostic or therapeutic elements. Combining computational approaches, we enriched miRNA binding motifs of Wnt pathway-associated upregulated genes. Our in-depth bioinformatics, in vitro and in vivo analyses indicated that miR-381 targets main genes of the Wnt signaling pathway including CTNNB1, RhoA, ROCK1, and c-MYC genes. The expression level of miR-381 and target genes was assessed by quantitative real-time polymerase chain reaction (RT-qPCR) in MCF-7, MDA-MB-231, and MCF-10A as well as 20 breast cancer samples and normal tissues. Luciferase reporter assay was performed. Lentiviral particles containing miR-381 were used to evaluate the effect of miR-381 restoration on cell proliferation, migration, and invasion of the invasive triple-negative MDA-MB-231 cell line and also in a mouse model of breast cancer. The expression of miR-381 was lower than that of normal cells, especially in TNBC cell line and breast tissues. Luciferase assay results confirmed that miR-381 targets all the predicted 3′-untranslated regions (3′-UTRs). Upon miR-381 overexpression, the expression of target genes declined, and the migration and invasion potential of miR-381-receiving MDA-MB-231 cells decreased. In a mouse model of triple-negative breast cancer, miR-381 re-expression inhibited the invasion of cancer cells to lung and liver and prolonged the survival time of cancer cell-bearing mice. Therefore, miR-381 is a regulator of Wnt signaling and its re-expression provides a potentially effective strategy for inhibition of TNBC.     

Silencing CDR1as enhances the sensitivity of breast cancer cells to drug resistance by acting as a miR‐7 sponge to down‐regulate REGγ

In our study, we aimed to investigate the role of CDR1as during competitive inhibition of miR‐7 in the regulation of cisplatin chemosensitivity in breast cancer via regulating REGγ. RT‐qPCR was applied to detect the expression of CDR1as and miR‐7 in breast cancer tissues, breast cancer cell lines and corresponding drug‐resistant cell lines. The correlation between CDR1as and miR‐7 and between miR‐7 and REGγ was evaluated. MCF‐7‐R and MDA‐MB‐231‐R cells were selected followed by transfection of a series of mimics, inhibitors or siRNA. The effect of CDR1as on the half maximal inhibitor concentration (IC50), cisplatin sensitivity and cell apoptosis was also analysed. Furthermore, a subcutaneous xenograft nude mouse model was established to further confirm the effect of CDR1as on the chemosensitivity of breast cancer to cisplatin in vivo. Immunohistochemical staining was conducted to test the Ki‐67 expression in nude mice. A positive correlation was found between the drug resistance and CDR1as expression in breast cancer. CDR1as could increase the resistance of breast cancer cells to cisplatin. miR‐7 expression was low, while REGγ was highly expressed in MCF‐7‐R and MDA‐MB‐231‐R cells. CDR1as competitively inhibited miR‐7 and up‐regulated REGγ. Overexpression of miR‐7 could reverse the enhanced sensitivity of silenced CDR1as to drug‐resistant breast cancer cells. Additionally, in vivo experiments demonstrated that CDR1as mediated breast cancer occurrence and its sensitivity to cisplatin. Silencing CDR1as decreased Ki‐67 expression. Silencing CDR1as may inhibit the expression of REGγ by removing the competitive inhibitory effect on miR‐7 and thus enhancing the sensitivity of drug‐resistant breast cancer cells.     

Inhibition of CDK-mediated Smad3 phosphorylation reduces the Pin1-Smad3 interaction and aggressiveness of triple negative breast cancer cells

Triple negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. Although TNBC is not defined by specific therapeutic targets, a subset of patients have tumors that overexpress cyclins. High cyclin D/E expression catalyzes CDK4/2 activity. In turn, CDK4/2 can non-canonically phosphorylate Smad3, a key TGFβ signaling intermediate, and this phosphorylation has been associated with the shift from tumor-suppressive to oncogenic TGFβ pathway action in breast oncogenesis. Additionally, CDK-mediated Smad3 phosphorylation facilitates an interaction between Smad3 and Pin1, a cis-trans isomerase that is also overexpressed in aggressive breast cancers. Treatment with CYC065, a CDK2/9 inhibitor, decreased non-canonical Smad3 phosphorylation and inhibited the Pin1-Smad3 interaction. We hypothesized that the interaction of Pin1 and Smad3, facilitated by CDK-mediated Smad3 phosphorylation, promotes TNBC cell aggressiveness. Inhibition of the Pin1-Smad3 interaction in TNBC cell lines, through depletion of Pin1 or CYC065 treatment, resulted in decreased cell migration/invasion and impeded the EMT program. Inhibition of CDK-mediated phosphorylation of Smad3 by mutagenesis also decreased cell migration, underscoring the importance of non-canonical CDK2 phosphorylation of Smad3 to enable cell motility. Pin1 depletion restored Smad3 protein levels and tumor-suppressive activity, suggesting that the Pin1-Smad3 interaction has a negative impact on canonical Smad3 action. Collectively, the data show that the Pin1-Smad3 interaction, facilitated by CDK-mediated Smad3 phosphorylation, is associated with oncogenic TGFβ signaling and breast cancer progression. Inhibition of this interaction with CYC065 treatment may provide an important therapeutic option for TNBC patients.     

The cyclin-dependent kinase inhibitor roscovitine and the nucleoside analog sangivamycin induce apoptosis in caspase-3 deficient breast cancer cells independent of caspase mediated P-glycoprotein cleavage: Implications for therapy of drug resistant breast cancers

Resistance to multiple chemotherapeutic agents is a common clinical problem which can arise during cancer treatment. Drug resistance often involves overexpression of the multidrug resistance MDR1 gene, encoding P-glycoprotein (P-gp), a 170-kDa glycoprotein belonging to the ATP-binding cassette superfamily of membrane transporters. We have recently demonstrated apoptosis-induced, caspase-3-dependent P-gp cleavage in human T-lymphoblastoid CEM-R VBL100 cells. However, P-gp contain many aspartate residues which could be targeted by caspases other than caspase-3. To test whether other caspases could cleave P-gp in vivo, we investigated the fate of P-gp during roscovitine- and sangivamycin- induced apoptosis in MCF7 human breast cancer cells, as they lack functional caspase-3. MCF7 cells were stably transfected with human cDNA encoding P-gp. P-gp was cleaved in vitro by purified recombinant caspase-3, -6 and -7. However, P-gp cleavage was not detected in vivo in MCF7 cells induced to undergoing apoptosis by either roscovitine or sangivamycin, despite activation of both caspase-6 and -7. Interestingly, P-gp overexpressing MCF7 cells were more sensitive to either roscovitine or sangivamycin than wild-type cells, suggesting a novel potential therapeutic strategy against P-gp overexpressing cells. Taken together, our results support the concept that caspase-3 is the only caspase responsible for in vivo cleavage of P-gp and also highlight small molecules which could be effective in treating P-gp overexpressing cancers.     

Regulation of breast cancer tumorigenesis and metastasis by miRNAs

miRNAs are a family of 17- to 23-nucleotide noncoding small RNAs that primarily function as gene expression fine regulators. A number of studies have shown that miRNAs play an important role in breast tumorigenesis, metastasis, proliferation and differentiation of breast cancer stem cells. This short review summarizes the progression of miRNA-mediated breast tumorigenesis and metastasis through various signaling pathways associated with drug resistance.     

Elevated CRB3 expression suppresses breast cancer stemness by inhibiting β‐catenin signalling to restore tamoxifen sensitivity

Tamoxifen is a first‐line drug for hormone therapy (HT) in oestrogen receptor‐positive breast cancer patients. However, 20% to 30% of those patients are resistant to tamoxifen treatment. Cancer stem cells (CSCs) have been implicated as one of the mechanisms responsible for tamoxifen resistance. Our previous study indicated that decreased expression of the CRB3 gene confers stem cell characteristics to breast cancer cells. In the current investigation, we found that most of the breast cancer patient tissues resistant to tamoxifen were negative for CRB3 protein and positive for β‐catenin protein, in contrast to their matched primary tumours by immunohistochemical analysis. Furthermore, expression of CRB3 mRNA and protein was low, while expression of β‐catenin mRNA and protein was high in tamoxifen resistance cells (LCC2 and T47D TamR) contrast to their corresponding cell lines MCF7 and T47D. Similarly, CRB3 overexpression markedly restored the tamoxifen sensitivity of TamR cells by the MTT viability assay. Finally, we found that CRB3 suppressed the stemness of TamR cells by inhibiting β‐catenin signalling, which may in turn lead to a decrease in the breast cancer cell population. Furthermore, these findings indicate that CRB3 is an important regulator for breast cancer stemness, which is associated with tamoxifen resistance.     

Synergistic anticancer action of quercetin and curcumin against triple-negative breast cancer cell lines

Women with the breast cancer type 1 susceptibility protein (BRCA1) mutation and loss of BRCA1 expression are reported to have an increased risk of triple-negative breast cancer (TNBC). Targeting BRCA1 modulation might offer a therapeutic option to treat TNBC patients. Our studies detected that BRCA1 is poorly expressed in TNBC cell lines and highly expressed in ER⁺ breast cancer cell lines. To modulate BRCA1 expression, we tested two different dietary components to find out if any would induce tumor suppressor genes. We detected that quercetin and curcumin dose-dependently enhanced the BRCA1 expression. Further, a synergistic action of quercetin and curcumin was observed in modulating the BRCA1 level and in inhibiting the cell survival and migration of TNBC cell lines. Quercetin and curcumin appeared to induce BRCA1 promoter histone acetylation. Furthermore, BRCA1 knockdown induced cell survival and cell migration in ER ⁺ cells were significantly decreased by the combined treatment of quercetin and curcumin. Our present study concluded that the combination treatment of quercetin and curcumin acts synergistically to induce anticancer activity against TNBC cells by modulating tumor suppressor genes.     

Effects of BCL-2 overexpression on the sensitivity of MCF-7 breast cancer cells to ricin, diphtheria and Pseudomonas toxin and immunotoxins

Immunotoxins are presently being evaluated as novel agents for cancer therapy. The direct mechanism by which immunotoxins kill cancer cells is inhibition of protein synthesis, but cytotoxicity due to induction of apoptosis has also been observed with these agents. Some cancers that express high levels of BCL-2 are relatively resistant to apoptosis inducing agents. It is therefore important to determine to what degree the toxicity of ricin, diphtheria toxin, Pseudomonas exotoxin and Pseudomonas exotoxin derived immunotoxins towards cancer cells can be attributed to inhibition of protein synthesis, and to what degree to subsequent induction of apoptosis. We compared the sensitivity of MCF-7 breast cancer cells that were stably transfected with a BCL-2 expression plasmid and thus protected against apoptosis and of MCF-7 cells transfected with a control plasmid towards ricin, diphtheria and Pseudomonas toxin, a Pseudomonas toxin-derived immunotoxin (LMB-7) and tumour necrosis factor (TNF). We found that BCL-2 mediated inhibition of apoptosis renders the cells almost completely resistant (1000-fold) to tumour necrosis factor, but the same cells were only 3–10 fold more resistant to cytotoxicity induced by immunotoxin LMB-7 as well as Pseudo-monas exotoxin, diphtheria toxin and ricin. We next studied several leukaemia cell lines with variable levels of BCL-2 expression and found them quite sensitive to a Pseudomonas exotoxin containing immunotoxin independent of the level of BCL-2. Our data indicate that although BCL-2 overexpression can have a modest effect on sensitivity to an immunotoxin, cell lines derived from patients are still very sensitive to immunotoxins.     

Human breast cancer histoid: an in vitro 3-dimensional co-culture model that mimics breast cancer tissue

Progress in our understanding of heterotypic cellular interaction in the tumor microenvironment, which is recognized to play major roles in cancer progression, has been hampered due to unavailability of an appropriate in vitro co-culture model. The aim of this study was to generate an in vitro 3-dimensional human breast cancer model, which consists of cancer cells and fibroblasts. Breast cancer cells (UACC-893) and fibroblasts at various densities were co-cultured in a rotating suspension culture system to establish co-culture parameters. Subsequently, UACC-893, BT.20, or MDA.MB.453 were co-cultured with fibroblasts for 9 days. Co-cultures resulted in the generation of breast cancer histoid (BCH) with cancer cells showing the invasion of fibroblast spheroids, which were visualized by immunohistochemical (IHC) staining of sections (4 μm thick) of BCH. A reproducible quantitative expression of C-erbB.2 was detected in UACC-893 cancer cells in BCH sections by IHC staining and the Automated Cellular Imaging System. BCH sections also consistently exhibited qualitative expression of pancytokeratins, p53, Ki-67, or E-cadherin in cancer cells and that of vimentin or GSTPi in fibroblasts, fibronectin in the basement membrane and collagen IV in the extracellular matrix. The expression of the protein analytes and cellular architecture of BCH were markedly similar to those of breast cancer tissue.