Adaptive resistance is not responsible for long-term drug resistance in a cellular model of triple negative breast cancer.

Resistance to cancer therapeutics represents a leading cause of mortality and is particularly important in cancers, such as triple negative breast cancer, for which no targeted therapy is available, as these are only treated with traditional chemotherapeutics. Cancer, as well as bacterial, drug resistance can be intrinsic, acquired or adaptive. Adaptive cancer drug resistance is gaining attention as a mechanism for the generation of long-term drug resistance as is the case with bacterial antibiotic resistance. We have used a cellular model of triple negative breast cancer (CAL51) and its drug resistance derivative (CALDOX) to gain insight into genome-wide expression changes associated with long-term doxorubicin (a widely used anthracycline for cancer treatment) resistance and doxorubicin-induced stress. Previous work indicates that both naïve and resistance cells have a functional p53-p21 axis controlling cell cycle at G1, although this is not a driver for drug resistance, but down-regulation of TOP2A (topoisomerase IIα). As expected, CALDOX cells have a signature characterized, in addition to down-regulation of TOP2A, by genes and pathways associated with drug resistance, metastasis and stemness. Both CAL51 and CALDOX stress signatures share 12 common genes (TRIM22, FAS, SPATA18, SULF2, CDKN1A, GDF15, MYO6, CXCL5, CROT, EPPK1, ZMAT3 and CD44), with roles in the above-mentioned pathways, indicating that these cells have similar functional responses to doxorubicin relaying on the p53 control of apoptosis. Eight genes are shared by both drug stress signatures (in CAL51 and CALDOX cells) and CALDOX resistant cells (FAS, SULF2, CDKN1A, CXCL5, CD44, SPATA18, TRIM22 and CROT), many of them targets of p53. This corroborates experimental data indicating that CALDOX cells, even in the absence of drug, have activated, at least partially, the p53-p21 axis and DNA damage response. Although this eight-gene signature might be an indicator of adaptive resistance, as this transient phenomenon due to short-term stress may not revert to its original state upon withdrawal of the stressor, previous experimental data indicates that the p53-p21 axis is not responsible for doxorubicin resistance. Importantly, TOP2A is not responsive to doxorubicin treatment and thus absent in both drug stress signatures. This indicates that during the generation of doxorubicin resistance, cells acquire genetic changes likely to be random, leading to down regulation of TOP2A, but selected during the generation of cells due to the presence of drug in the culture medium. This poses a considerable constraint for the development of strategies aimed at avoiding the emergence of drug resistance in the clinic.

MicroRNA-495/TGF-ß/FOXC1 axis regulates multidrug resistance in metaplastic breast cancer cells.

Triple-negative metaplastic breast carcinoma (MBC) poses a significant treatment challenge due to lack of targeted therapies and chemotherapy resistance. We isolated a novel MBC cell line, BAS, which showed a molecular and phenotypic profile different from the only other metaplastic cell model, HS578T cells. To gain insight behind chemotherapeutic resistance, we generated doxorubicin (HS-DOX, BAS-DOX) and paclitaxel (HS-TX, BAS-TX) resistant derivatives of both cell lines. Drug sensitivity assays indicated a truly multidrug resistant (MDR) phenotype. Both BAS-DOX and BAS-TX showed up-regulation of FOXC1 and its experimental down-regulation re-sensitized cells to doxorubicin and paclitaxel. Experimental modulation of FOXC1 expression in MCF-7 and MDA-MB-231 cells corroborated its role in MDR. Genome-wide expression analyses identified gene expression signatures characterized by up-regulation of TGFB2, which encodes cytokine TGF-ß2, in both BAS-DOX and BAS-TX cells. Pharmacological inhibition of the TGF-ß pathway with galunisertib led to down-regulation of FOXC1 and increase in drug sensitivity in both BAS-DOX and BAS-TX cells. MicroRNA (miR) expression analyses identified high endogenous miR-495-3p levels in BAS cells that were downregulated in both BAS MDR cells. Transient expression of miR-495-3p mimic in BAS-DOX and BAS-TX cells caused downregulation of TGFB2 and FOXC1 and re-sensitized cells to doxorubicin and paclitaxel, whereas miR-495-3p inhibition in BAS cells led to increase in resistance to both drugs and up-regulation of TGFB2 and FOXC1. Together, these data suggest interplay between miR-495-3p, TGF-ß2 and FOXC1 regulating MDR in MBC and open the exploration of novel therapeutic strategies.

FOXA1 is a determinant of drug resistance in breast cancer cells.

PURPOSE: Breast cancer is one of the most commonly diagnosed cancers in women. Five subtypes of breast cancer differ in their genetic expression profiles and carry different prognostic values, with no treatments available for some types, such as triple-negative, due to the absence of genetic signatures that could otherwise be targeted by molecular therapies. Although endocrine treatments are largely successful for estrogen receptor (ER)-positive cancers, a significant proportion of patients with metastatic tumors fail to respond and acquire resistance to therapy. FOXA1 overexpression mediates endocrine therapy resistance in ER-positive breast cancer, although the regulation of chemotherapy response by FOXA1 has not been addressed previously. FOXA1, together with EP300 and RUNX1, regulates the expression of E-cadherin, and is expressed in luminal, but absent in triple-negative and basal-like breast cancers. We have previously determined that EP300 regulates drug resistance and tumor initiation capabilities in breast cancer cells. METHODS: Here we describe the generation of breast cancer cell models in which FOXA1 expression has been modulated either by expression of hairpins targeting FOXA1 mRNA or overexpression plasmids. RESULTS: Upon FOXA1 knockdown in luminal MCF-7 and T47D cells, we found an increase in doxorubicin and paclitaxel sensitivity as well as a decrease in anchorage independence. Conversely, upregulation of FOXA1 in basal-like MDA-MB-231 cells led to an increase in drug resistance and anchorage independence. CONCLUSION: Together, these data suggest that FOXA1 plays a role in making tumors more aggressive.

Oncogenic EP300 can be targeted with inhibitors of aldo-keto reductases.

E-cadherin transcriptional activator EP300 is down-regulated in metaplastic breast carcinoma, a rare form of triple negative and E-cadherin-negative aggressive breast cancer with a poor clinical outcome. In order to shed light on the regulation of E-cadherin by EP300 in breast cancer we analyzed by immunohistochemistry 41 cases of invasive breast cancer with both E-cadherinhigh and E-cadherinlow expression levels, together with 20 non-malignant breast tissues. EP300 and E-cadherin showed a positive correlation in both non-malignant and cancer cases and both markers together were better predictors of lymph node metastasis than E-cadherin alone. These data support a metastasis suppressor role for EP300 in breast cancer. However, some reports suggest an oncogenic role for EP300. We generated a breast cancer cell model to study E-cadherin-independent effects of EP300 by over-expression of EP300 in HS578T cells which have E-cadherin promoter hypermethylated. In this cell system, EP300 led to up-regulation of mesenchymal (vimentin, Snail, Slug, Zeb1) and stemness (ALDH+ and CD44high/CD24low) markers, increases in migration, invasion, anchorage-independent growth and drug resistance. Genome-wide expression profiling identified aldo-keto reductases AKR1C1-3 as effectors of stemness and drug resistance, since their pharmacological inhibition with flufenamic acid restored both doxorubicin and paclitaxel sensitivity and diminished mammosphere formation. Thus, in cells with a permissive E-cadherin promoter, EP300 acts as a tumour/metastasis supressor by up-regulating E-cadherin expression, maintenance of the epithelial phenotype and avoidance of an epithelial-to-mesenchymal transition. In cells in which the E-cadherin promoter is hypermethylated, EP300 functions as an oncogene via up-regulation of aldo-keto reductases. This offers the rationale of using current aldo-keto reductase inhibitors in breast cancer treatment.