in breast cancer

PSMD9 expression predicts radiotherapy respons

MiR-195 and Its Target SEMA6D Regulate Chemoresponse in Breast Cancer

From MDPI via Jisc Publications RouterHistory: accepted 2021-11-26, pub-electronic 2021-11-28Publication status: PublishedBackground: 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

Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial.

Aims Mutation detection accuracy has been described extensively; however, it is surprising that pre-PCR processing of formalin-fixed paraffin-embedded (FFPE) samples has not been systematically assessed in clinical context. We designed a RING trial to (i) investigate pre-PCR variability, (ii) correlate pre-PCR variation with EGFR/BRAF mutation testing accuracy and (iii) investigate causes for observed variation. Methods 13 molecular pathology laboratories were recruited. 104 blinded FFPE curls including engineered FFPE curls, cell-negative FFPE curls and control FFPE tissue samples were distributed to participants for pre-PCR processing and mutation detection. Follow-up analysis was performed to assess sample purity, DNA integrity and DNA quantitation. Results Rate of mutation detection failure was 11.9%. Of these failures, 80% were attributed to pre-PCR error. Significant differences in DNA yields across all samples were seen using analysis of variance (p<0.0001), and yield variation from engineered samples was not significant (p=0.3782). Two laboratories failed DNA extraction from samples that may be attributed to operator error. DNA extraction protocols themselves were not found to contribute significant variation. 10/13 labs reported yields averaging 235.8ng (95% CI 90.7 to 380.9) from cell-negative samples, which was attributed to issues with spectrophotometry. DNA measurements using Qubit Fluorometry demonstrated a median fivefold overestimation of DNA quantity by Nanodrop Spectrophotometry. DNA integrity and PCR inhibition were factors not found to contribute significant variation. Conclusions In this study, we provide evidence demonstrating that variation in pre-PCR steps is prevalent and may detrimentally affect the patient's ability to receive critical therapy. We provide recommendations for preanalytical workflow optimisation that may reduce errors in down-stream sequencing and for next-generation sequencing library generation

Neoadjuvant Endocrine Therapy in Breast Cancer Upregulates the Cytotoxic Drug Pump ABCG2/BCRP, and May Lead to Resistance to Subsequent Chemotherapy

Introduction: Neoadjuvant treatments for primary breast cancer are becoming more common; however, little is known about how these impact on response to subsequent adjuvant therapies. Conveniently, neoadjuvant therapy provides opportunities to consider this question, by studying therapy-induced expression changes using comparisons between pre- and posttreatment samples. These data are relatively lacking in the context of neoadjuvant endocrine therapy, as opposed to the more common neoadjuvant chemotherapy. Here, we investigate the relevance of expression of the xenobiotic transporter ABCG2/BCRP, a gene/protein associated with chemoresistance, in the context of neoadjuvant endocrine therapy and particularly with reference to subsequent chemotherapy treatment. Materials and Methods: ABCG2/BCRP expression was assessed by immunohistochemistry or by expression arrays in matched patient samples pre- and post-neoadjuvant endocrine therapy. Cell culture was used to model the impact of endocrine therapy-induced changes in ABCG2/BCRP on subsequent chemotherapy response, using Western blots, quantitative polymerase chain reaction, survival assays, and cell cycle analyses. Results: ABCG2/BCRP was commonly and significantly upregulated in breast cancers after treatment with neoadjuvant endocrine therapy in 3 separate cohorts encompassing a total of 200 patients. Treatment with the endocrine therapeutic tamoxifen similarly induced ABCG2/BCRP upregulation in a relevant model cell line, the estrogen receptor-positive line T47D. Critically, this upregulation was associated with significantly increased chemoresistance to subsequent treatment with epirubicin, an anthracycline commonly used in breast cancer adjuvant chemotherapy. Conclusion: Our data suggest that neoadjuvant endocrine therapy may induce poor responses to adjuvant chemotherapy, and therefore, that clinical outcomes following this treatment sequence warrant further study

Chemotherapy induces Notch1-dependent MRP1 up-regulation, inhibition of which sensitizes breast cancer cells to chemotherapy

Background Multi-drug Resistance associated Protein-1 (MRP1) can export chemotherapeutics from cancer cells and is implicated in chemoresistance, particularly as is it known to be up-regulated by chemotherapeutics. Our aims in this study were to determine whether activation of Notch signalling is responsible for chemotherapy-induced MRP1 expression Notch in breast cancers, and whether this pathway can be manipulated with an inhibitor of Notch activity. Methods MRP1 and Notch1 were investigated in 29 patients treated with neoadjuvant chemotherapy (NAC) for breast cancer, using immunohistochemistry on matched biopsy (pre-NAC) and surgical samples (post-NAC). Breast epithelial cell cultures (T47D, HB2) were treated with doxorubicin in the presence and absence of functional Notch1, and qPCR, siRNA, Western blots, ELISAs and flow-cytometry were used to establish interactions. Results In clinical samples, Notch1 was activated by neoadjuvant chemotherapy (Wilcoxon signed-rank p < 0.0001) and this correlated with induction of MRP1 expression (rho = 0.6 p = 0.0008). In breast cell lines, doxorubicin induced MRP1 expression and function (non-linear regression p < 0.004). In the breast cancer line T47D, doxorubicin activated Notch1 and, critically, inhibition of Notch1 activation with the γ-secretase inhibitor DAPT abolished the doxorubicin-induced increase in MRP1 expression and function (t-test p < 0.05), resulting in enhanced cellular retention of doxorubicin and increased doxorubicin-induced apoptosis (t-test p = 0.0002). In HB2 cells, an immortal but non-cancer derived breast cell line, Notch1-independent MRP1 induction was noted and DAPT did not enhance doxorubicin-induced apoptosis. Conclusions Notch inhibitors may have potential in sensitizing breast cancer cells to chemotherapeutics and therefore in tackling chemoresistance

Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis

Background MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR–92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR–92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR–92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells. Methodology/Principal Findings We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR–92 expression by qRT-PCR. Expression of ERβ1, a direct miR–92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR–92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR– 92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERβ1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR–92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR–92 levels in NFs but not CAFs enhanced invasion of both MCF–7 and MDA-MB–231 breast cancer epithelial cells. Conclusions miR–92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERβ1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR–92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERβ1 may not be the most important miR–92 target in breast cancer

Epithelial stromal interaction in breast cancer

In recent years there has been an increasing awareness of the role played by the microenvironment surrounding breast cancer epithelium in modulating the behaviour of breast tumours. A major component of this microenvironment in breast cancer is the fibroblasts. These are commonly referred to as carcinomaassociated fibroblasts (CAFs). CAFs are phenotypically different from their normal counterpart in normal breast tissue. MicroRNAs, a family of small noncoding RNAs that are key players in the posttranscriptional regulation of mRNAs, have been shown to play a role in controlling the behaviour of cancers. They have been extensively studied in breast cancer epithelial cells and have important roles in breast cancer initiation and progression. My hypotheses were that miRNAs also play roles in controlling the behaviour of breast CAFs and in turn, impact on the behaviour of malignant breast epithelial cells. I used a single patient sample and a co-culture model to examine the expression of miRNAs in breast CAFs and normal fibroblast (NFs). Six miRNAs were consistently down-regulated in CAFs as compared to NFs with a fold change > 10 in both the tissue co-culture model and in patient samples. Of these, miR-26b was significantly down-regulated in CAFs in a further 14 cases of microdissected matched NFs and CAFs from clinical FFPE (Formalin Fixed Paraffin Embedded) samples and consistently in a further 4 cases of matched primary NFs and CAFs in vitro. Functional influences on fibroblasts were examined by overexpressing and ‘knocking down’ miR-26b. Although overexpression of miR-26 in immortalised breast fibroblasts was associated with decreased proliferation and increased apoptosis, this was felt to be not physiologically relevant since the degree of over-expression exceeded the differential expression seen in clinical samples. Reducing the level of miR-26b in immortalised breast fibroblasts resulted in a small decrease in proliferation, and a very notable increase in migration and invasion. Next, the functional influences of fibroblasts with reduced miR-26b on breast cancer epithelial cells were examined. There was no difference in the growth of cancer epithelial cells when they were co-cultured with fibroblasts with low miR-26b as compared to controls. However, these fibroblasts increased the migration and invasion of breast cancer epithelial cells. To identify the potential targets and pathways targeted by miR-26b, I used a combination of mass spectrometry and in-silico analysis. I identified multiple proteins that were differentially expressed between miR-26b knockdown and control fibroblasts. COL12-A1, TNKS1BP1 and CPSF7 were identified as potential targets of miR-26b. I used publically available databases to examine the clinical relevance of these targets in the stroma of breast cancers. Higher expression of these targets was associated with increased rates of recurrence of breast cancer. In addition, pathway analyses of the differentially expressed proteins were enriched for components of the “glycolytic” pathway and “cytoskeleton regulation by RHO GTPase” pathway. In conclusion, I have shown that down regulation of miR-26b in breast cancer fibroblasts increases the migration and invasion of breast cancer epithelial cells, and I have identified molecular changes that are downstream of miR-26b

Differential regulation of oestrogen receptor β isoforms by 5′ untranslated regions in cancer

Oestrogen receptors (ERs) are critical regulators of the behaviour of many cancers. Despite this, the roles and regulation of one of the two known ERs – ERβ– are poorly understood. This is partly because analyses have been confused by discrepancies between ERβ expression at mRNA and proteins levels, and because ERβ is expressed as several functionally distinct isoforms. We investigated human ERβ 5′ untranslated regions (UTRs) and their influences on ERβ expression and function. We demonstrate that two alternative ERβ 5′UTRs have potent and differential influences on expression acting at the level of translation. We show that their influences are modulated by cellular context and in carcinogenesis, and demonstrate the contributions of both upstream open reading frames and RNA secondary structure. These regulatory mechanisms offer explanations for the non-concordance of ERβ mRNA and protein. Importantly, we also demonstrate that 5′UTRs allow the first reported mechanisms for differential regulation of the expression of the ERβ isoforms 1, 2 and 5, and thereby have critical influences on ERβ function

Neoadjuvant Chemotherapy Induces Expression Levels of Breast Cancer Resistance Protein That Predict Disease-Free Survival in Breast Cancer

<div><p>Three main xenobiotic efflux pumps have been implicated in modulating breast cancer chemotherapy responses. These are P-glycoprotein (Pgp), Multidrug Resistance-associated Protein 1 (MRP1), and Breast Cancer Resistance Protein (BCRP). We investigated expression of these proteins in breast cancers before and after neoadjuvant chemotherapy (NAC) to determine whether their levels define response to NAC or subsequent survival. Formalin-fixed paraffin-embedded tissues were collected representing matched pairs of core biopsy (pre-NAC) and surgical specimen (post-NAC) from 45 patients with invasive ductal carcinomas. NAC regimes were anthracyclines +/− taxanes. Immunohistochemistry was performed for Pgp, MRP1 and BCRP and expression was quantified objectively using computer-aided scoring. Pgp and MRP1 were significantly up-regulated after exposure to NAC (Wilcoxon signed-rank p = 0.0024 and p<0.0001), while BCRP showed more variation in response to NAC, with frequent up- (59% of cases) and down-regulation (41%) contributing to a lack of significant difference overall. Pre-NAC expression of all markers, and post-NAC expression of Pgp and MRP1 did not correlate with NAC response or with disease-free survival (DFS). Post-NAC expression of BCRP did not correlate with NAC response, but correlated significantly with DFS (Log rank p = 0.007), with longer DFS in patients with low post-NAC BCRP expression. In multivariate Cox regression analyses, post-NAC BCRP expression levels proved to predict DFS independently of standard prognostic factors, with high expression associated with a hazard ratio of 4.04 (95% confidence interval 1.3–12.2; p = 0.013). We conclude that NAC-induced expression levels of BCRP predict survival after NAC for breast cancer, while Pgp and MRP1 expression have little predictive value.</p></div