Background Cancer-associated Þbroblasts (CAFs) are known to impact on tumour behaviour but mechanisms controlling this are poorly understood. Methods Breast normal fibroblasts (NFs) or CAFs were isolated from cancers by laser-microdissection or were cultured. Fibroblasts were transfected to manipulate miR-222 or Lamin B Receptor (LBR). Fibroblast conditioned medium was collected and used to treat epithelial BC lines MDA-MB-231 and MDA-MB-157. Migration, invasion, proliferation, or senescence was assessed using transwell, MTT or X-gal assays respectively. Results MiR-222 was up-regulated in CAFs as compared to NFs. Ectopic miR-222 expression in NFs induced CAF-like expression profiles, while miR-222 knock-down in CAFs inhibited CAF phenotypes. LBR was identified as a direct miR-222 target and was functionally relevant since LBR knock-down phenocopied miR-222 over-expression and LBR over-expression phenocopied miR-222 knock-down. MiR-222 over-expression, or LBR knock-down, was sufficient to induce NFs to show CAF characteristics of enhanced migration, invasion and senescence, and furthermore conditioned medium from these fibroblasts induced increased BC cell migration and invasion. The reverse manipulations in CAFs inhibited these behaviours in fibroblasts and inhibited paracrine influences on BC cells. Conclusion MiR-222/LBR have key roles in controlling pro-progression influences of CAFs in BC. This pathway may present therapeutic opportunities to inhibit CAF-induced cancer progression. 3 BACKGROUND Breast cancer (BC) is the leading cause of cancer death worldwide among women 1 , and nearly 2.3 million females are newly diagnosed annually. Although there are initial responses to treatment, many cancers relapse and distant metastases occur in nearly one third of woman; these are typically fatal. The biology behind BC metastases still remains undetermined. Therefore, understanding molecular determinants of metastasis is crucial for finding new therapeutic strategies. The tumour microenvironment consists of immune cells, blood vessels, endothelial cells, fibroblasts and extracellular matrix 4. This microenvironment plays key roles in disease outcome by inducing tumour cell proliferation and aggressiveness 5,6. Cancer-associated Þbroblasts (CAFs), an activated form of tissue-resident fibroblasts present within breast cancers, comprise a major component of the tumour microenvironment 7 , characterised most commonly by expression of-smooth muscle actin 8. CAFs can induce cancer progression 9 and metastasis 10 by secreting various cytokines, chemokines and growth factors (e.g. VEGF, FGF2, TGF , CXCL12, IL6 and IL8) 11,12 and by modulating the extracellular matrix (ECM) that facilitates tumour cell migration and invasion 13,14. CAFs also modulate immune cell function to create an immune suppressive environment during cancer progression 15. The process of transformation of CAFs from resident normal Þbroblasts is achieved by several growth factors 16,17,18 , however mechanisms of transformation have not yet been fully expl...

This is a repository copy of Inhibition of interferon-signalling halts cancer-associated fibroblast-dependent protection of breast cancer cells from chemotherapy.

This is a repository copy of MiR-19b non-canonical binding is directed by HuR and confers chemosensitivity through regulation of P-glycoprotein in breast cancer.

Triple negative breast cancer (TNBC) is challenging to treat successfully because targeted therapies do not exist. Instead, systemic therapy is typically restricted to cytotoxic chemotherapy, which fails more often in patients with elevated circulating cholesterol. Liver x receptors are ligand-dependent transcription factors that are homeostatic regulators of cholesterol, and are linked to regulation of broad-affinity xenobiotic transporter activity in non-tumor tissues. We show that LXR ligands confer chemotherapy resistance in TNBC cell lines and xenografts, and that LXRalpha is necessary and sufficient to mediate this resistance. Furthermore, in TNBC patients who had cancer recurrences, LXRalpha and ligands were independent markers of poor prognosis and correlated with P-glycoprotein expression. However, in patients who survived their disease, LXRalpha signaling and P-glycoprotein were decoupled. These data reveal a novel chemotherapy resistance mechanism in this poor prognosis subtype of breast cancer. We conclude that systemic chemotherapy failure in some TNBC patients is caused by co-opting the LXRalpha:P-glycoprotein axis, a pathway highly targetable by therapies that are already used for prevention and treatment of other diseases.

Triple negative breast cancer (TNBC) is challenging to treat successfully because targeted therapies do not exist. Instead, systemic therapy is typically restricted to cytotoxic chemotherapy, which fails more often in patients with elevated circulating cholesterol. Liver x receptors are ligand-dependent transcription factors that are homeostatic regulators of cholesterol, and are linked to regulation of broad-affinity xenobiotic transporter activity in non-tumor tissues. We show that LXR ligands confer chemotherapy resistance in TNBC cell lines and xenografts, and that LXRalpha is necessary and sufficient to mediate this resistance. Furthermore, in TNBC patients who had cancer recurrences, LXRalpha and ligands were independent markers of poor prognosis and correlated with P-glycoprotein expression. However, in patients who survived their disease, LXRalpha signaling and P-glycoprotein were decoupled. These data reveal a novel chemotherapy resistance mechanism in this poor prognosis subtype of breast cancer. We conclude that systemic chemotherapy failure in some TNBC patients is caused by co-opting the LXRalpha:P-glycoprotein axis, a pathway highly targetable by therapies that are already used for prevention and treatment of other diseases.

CIP2A is emerging as an oncoprotein overexpressed commonly across many tumours and generally correlated with higher tumour grade and therapeutic resistance. CIP2A drives an oncogenic potential through inhibiting protein phosphatase 2A, stabilizing MYC, and promoting epithelial-to-mesenchymal transition, although further biological mechanisms for CIP2A are yet to be defined. CIP2A protein expression was studied by immunohistochemistry in oestrogen receptor-positive primary breast cancers (n = 250) obtained from the Leeds Tissue Bank. In total, 51 cases presented with a relapse or metastasis during adjuvant treatment with tamoxifen and were regarded as tamoxifen resistant. CIP2A expression was scored separately for cytoplasmic, nuclear, or membranous staining, and scores were tested for statistically significant relationships with clinicopathological features. Membranous CIP2A was preferentially expressed in cases who experienced a recurrence during tamoxifen treatment thus predicting a worse overall survival (log rank = 8.357, p = 0.004) and disease-free survival (log rank = 21.766, p < 0.001). Cox multivariate analysis indicates that it is an independent prognostic indicator for overall survival (hazard ratio = 4.310, p = 0.013) and disease-free survival (hazard ratio = 5.449, p = 0.002). In this study, we propose the assessment of membranous CIP2A expression as a potential novel prognostic and predictive indicator for tamoxifen resistance and recurrence within oestrogen receptor-positive breast cancer.

Although the UK National Institute for Health and Care Excellence guidelines recommend that in patients with biopsy-proven invasive lobular carcinoma (ILC), preoperative MRI scan is considered, the accuracy of diagnosis of ILC in core biopsy of the breast has not been previously investigated. Eleven pathology laboratories from the UK and Ireland submitted data on 1112 cases interpreted as showing features of ILC, or mixed ILC and IDC/no special type (NST)/other tumour type, on needle core biopsy through retrieval of histology reports. Of the total 1112 cases, 844 were shown to be pure ILC on surgical excision, 154 were mixed ILC plus another type (invariably ductal/NST) and 113 were shown to be ductal/NST. Of those lesions categorised as pure ILC on core, 93% had an element of ILC correctly identified in the core biopsy sample and could be considered concordant. Of cores diagnosed as mixed ILC plus another type on core, complete agreement between core and excision was 46%, with 27% cases of pure ILC, whilst 26% non-concordant. These data indicate that there is not a large excess of expensive MRIs being performed as a result of miscategorisation histologically.

Triple negative breast cancers (TNBCs) lack cellular protein receptors that can be targeted by the most effective breast cancer drugs. Instead, these patients are treated with non-specific, systemic, cytotoxic chemotherapy agents, such as epirubicin, which can cause severe side effects. Furthermore, chemotherapy resistance is common, rendering many treatments ineffective. P-glycoprotein (Pgp) is a chemotherapy efflux pump that is commonly over-expressed in chemoresistant tumours. However, pharmacological inhibitors of Pgp have typically failed in trials owing to Pgp's crucial role in other tissues; tumour specific mechanisms of Pgp regulation should instead be targeted to induce chemosensitization. Pgp is regulated by cholesterol side-chain hydroxylation products (scOHC) and the liver x receptor (LXR) in the blood brain barrier (1) , potentially linking nutrition and cholesterol metabolism to cellular drug efflux. Interestingly, breast cancer relapse is associated with diets that drive elevated levels of LXR ligands via high circulating cholesterol (2) . Our aim was to determine if expression of LXR and synthesis of its scOHC ligands was associated and/or causative of i) elevated P-gp expression in TNBC, and ii) poor disease-free survival of TNBC patients.Cancer cell expression of Pgp and scOHC synthesising enzymes (CYP46A1, CH25H, CYP27A1) was assessed with immunohistochemistry (as previously (3) ) using a tissue micro-array containing tissue from 148 TNBC tumours collected at Leeds Teaching Hospitals Trust (ethics reference 06/Q1206/180). In a separate cohort of 31 fresh/frozen TNBC tumours (ethics reference 15/HY/ 0025), we determined: scOHC concentrations for 24OHC, 25OHC, and 26OHC (also known as 27OHC) using LC-MS/MS (4) ; and Pgp mRNA expression using TaqMan-qPCR (DDcT). In vitro, TNBC cell lines (MDA.MBA.231 and MBA.MB.468) were treated with scOHCs and changes to Pgp mRNA expression assessed by TaqMan. Epirubicin efficacy after scOHC exposure was determined with colony forming assays and MTT. Spearman rank correlation, log-rank Kaplan Meier plots, and one-way ANOVA were used for statistical analysis.Pgp protein expression in cancer cells in primary tumours positively correlated with CYP46A1 (R 2 =0.3; p < 0.0001), CH25H (R 2 =0.53; p < 0.0001), and CYP27A1 (R 2 =0.07; p < 0.01). High levels of P-gp, CYP46A1 and CH25H (log-rank test: p < 0.01 for all) were all associated with reduced disease-free survival. With respect to the scOHCs, 24OHC correlated with P-gp mRNA (R 2 =0.5; p = 0.04), but only elevated 25OHC and 26OHC associated with reduced disease-free survival (p < 0.05). In vitro, addition of 24OHC, 25OHC or 26OHC to TNBC cell lines increased P-gp mRNA and reduced epirubicin mediated cytotoxicity in TNBC cells (p < 0.001).These results suggest that high intratumour oxysterol content is linked to reduced disease-free survival, potentially due to enhanced chemotherapy resistance via Pgp mediated drug efflux. Nutritional and pharmacological reduction of oxysterol levels should be explored as routes to r...