Abstract
Simple SummaryThe resistance of cancer cells to cytotoxic chemotherapy limits cure rates in breast cancer, and a better understanding of resistance mechanisms will aid design of strategies to improve treatments. We have identified molecules that cause chemoresistance through comparison of matched breast cancer samples from before and after chemotherapy treatment, and by manipulating their levels in cultured breast cancer cells and measuring chemotherapy-induced cell death. We show that microRNA-195 and microRNA-26b induce resistance to chemotherapy in breast cancer by reducing the levels of the protein SEMA6D. Accordingly, levels of SEMA6D in breast cancers predict the survival of patients after chemotherapy. SEMA6D is a predictive marker and SEMA6D signaling presents a therapeutic opportunity for sensitizing cells to chemotherapy.Background: poor prognosis primary breast cancers are typically treated with cytotoxic chemotherapy. However, recurrences remain relatively common even after this aggressive therapy. Comparison of matched tumours pre- and post-chemotherapy can allow identification of molecular characteristics of therapy resistance and thereby potentially aid discovery of novel predictive markers or targets for chemosensitisation. Through this comparison, we aimed to identify microRNAs associated with chemoresistance, define microRNA target genes, and assess targets as predictors of chemotherapy response. Methods: cancer cells were laser microdissected from matched breast cancer tissues pre- and post-chemotherapy from estrogen receptor positive/HER2 negative breast cancers showing partial responses to epirubicin/cyclophosphamide chemotherapy (n = 5). MicroRNA expression was profiled using qPCR arrays. MicroRNA/mRNA expression was manipulated in estrogen receptor positive/HER2 negative breast cancer cell lines (MCF7 and MDA-MB-175 cells) with mimics, inhibitors or siRNAs, and chemoresponse was assessed using MTT and colony forming survival assays. MicroRNA targets were identified by RNA-sequencing of microRNA mimic pull-downs, and comparison of these with mRNAs containing predicted microRNA binding sites. Survival correlations were tested using the METABRIC expression dataset (n = 1979). Results: miR-195 and miR-26b were consistently up-regulated after therapy, and changes in their expression in cell lines caused significant differences in chemotherapy sensitivity, in accordance with up-regulation driving resistance. SEMA6D was defined and confirmed as a target of the microRNAs. Reduced SEMA6D expression was significantly associated with chemoresistance, in accordance with SEMA6D being a down-stream effector of the microRNAs. Finally, low SEMA6D expression in breast cancers was significantly associated with poor survival after chemotherapy, but not after other therapies. Conclusions: microRNAs and their targets influence chemoresponse, allowing the identification of SEMA6D as a predictive marker for chemotherapy response that could be used to direct therapy or as a target in chemosensitisation strategies.
Highlights
Cytotoxic chemotherapy is a component of treatment for around a third of primary breast cancers, comprising those associated with relatively poor prognoses such as larger, lymph node positive, HER2-positive, or triple-negative tumours [1]
Cancers 2021, 13, 5979 containing all the diversity of breast cancer subtypes that are treated with chemotherapy, demonstrating its applicability to this treatment modality beyond the estrogen receptor positive/HER2 negative subtype used for discovery
Patients included were diagnosed within LTH NHS Trust (Leeds, UK) with estrogen receptor (ER) positive/HER2 negative primary invasive ductal breast carcinoma and were treated with 6 cycles of neoadjuvant epirubicin/cyclophosphamide, showing partial treatment resistance as determined by longitudinal MRI imaging and histopathology assessments of resections
Summary
Cytotoxic chemotherapy is a component of treatment for around a third of primary breast cancers, comprising those associated with relatively poor prognoses such as larger, lymph node positive, HER2-positive, or triple-negative tumours [1]. Neoadjuvant chemotherapy provides the further advantage that matched tumour tissue from pre- and post-therapy can be available, allowing for the comparative identification of the therapy-associated changes within the cancer cells that may be associated with relative chemotherapy resistance. The discovery of potential cancer cell autonomous resistance mechanisms through comparisons of whole tissue samples from pre- and post-therapy can be confounded by differences in tissue composition This issue has previously been addressed by the exclusion of mRNAs for stromal-associated genes from any findings [11]. Cancers 2021, 13, 5979 containing all the diversity of breast cancer subtypes that are treated with chemotherapy, demonstrating its applicability to this treatment modality beyond the estrogen receptor positive/HER2 negative subtype used for discovery
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