Rebecca Millican-Slater

The sodium channel β1 subunit mediates outgrowth of neurite-like processes on breast cancer cells and promotes tumour growth and metastasis

Voltage‐gated Na+ channels (VGSCs) are heteromeric proteins composed of pore‐forming α subunits and smaller β subunits. The β subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). β1, encoded by SCN1B, is best characterized in the central nervous system (CNS), where it plays a critical role in regulating electrical excitability, neurite outgrowth and migration during development. β1 is also expressed in breast cancer (BCa) cell lines, where it regulates adhesion and migration in vitro. In the present study, we found that SCN1B mRNA/β1 protein were up‐regulated in BCa specimens, compared with normal breast tissue. β1 upregulation substantially increased tumour growth and metastasis in a xenograft model of BCa. β1 over‐expression also increased vascularization and reduced apoptosis in the primary tumours, and β1 over‐expressing tumour cells had an elongate morphology. In vitro, β1 potentiated outgrowth of processes from BCa cells co‐cultured with fibroblasts, via trans‐homophilic adhesion. β1‐mediated process outgrowth in BCa cells required the presence and activity of fyn kinase, and Na+ current, thus replicating the mechanism by which β1 regulates neurite outgrowth in CNS neurons. We conclude that when present in breast tumours, β1 enhances pathological growth and cellular dissemination. This study is the first demonstration of a functional role for β1 in tumour growth and metastasis in vivo. We propose that β1 warrants further study as a potential biomarker and targeting β1‐mediated adhesion interactions may have value as a novel anti‐cancer therapy.

The practicalities of using tissue slices as preclinical organotypic breast cancer models

Models considering breast cancer complexity cannot be easily or accurately replicated in routine cell line or animal models. We aimed to evaluate the practicality of organotypic tissue slice culture in breast cancer. Following ethical approval, 250 µm thick sections from surplus breast tumours (n=10) were prepared using a vibrating blade microtome. Triplicate tissue slices were placed in 6-well plates and cultured for up to 7 days±tamoxifen (1 nM) or doxorubicin (1 µM). Tissue slices were fixed and embedded before sectioning for morphological evaluation and immunohistochemistry. H&E showed good preservation of tissue morphology. Collagen production was evident. Biomarkers of proliferation and apoptosis could be evaluated using immunohistochemistry and used as surrogates to quantify drug effects. In summary, breast cancer tissue slices can be cultured in vitro as organotypic models. Nevertheless, although simple in concept, the delicacy of the model with regard to handling makes subsequent analytical processes challenging.

Nav1.5 regulates breast tumor growth and metastatic dissemination in vivo.

Voltage-gated Na+ channels (VGSCs) mediate action potential firing and regulate adhesion and migration in excitable cells. VGSCs are also expressed in cancer cells. In metastatic breast cancer (BCa) cells, the Nav1.5 α subunit potentiates migration and invasion. In addition, the VGSC-inhibiting antiepileptic drug phenytoin inhibits tumor growth and metastasis. However, the functional activity of Nav1.5 and its specific contribution to tumor progression in vivo has not been delineated. Here, we found that Nav1.5 is up-regulated at the protein level in BCa compared with matched normal breast tissue. Na+ current, reversibly blocked by tetrodotoxin, was retained in cancer cells in tumor tissue slices, thus directly confirming functional VGSC activity in vivo. Stable down-regulation of Nav1.5 expression significantly reduced tumor growth, local invasion into surrounding tissue, and metastasis to liver, lungs and spleen in an orthotopic BCa model. Nav1.5 down-regulation had no effect on cell proliferation or angiogenesis within the in tumors, but increased apoptosis. In vitro, Nav1.5 down-regulation altered cell morphology and reduced CD44 expression, suggesting that VGSC activity may regulate cellular invasion via the CD44-src-cortactin signaling axis. We conclude that Nav1.5 is functionally active in cancer cells in breast tumors, enhancing growth and metastatic dissemination. These findings support the notion that compounds targeting Nav1.5 may be useful for reducing metastasis.

Outcome of a new patient pathway for managing B3 breast lesions by vacuum-assisted biopsy: time to change current UK practice?

Aims B3 lesions of the breast represent a difficult management dilemma. The umbrella term ‘B3’ incorporates lesions with little associated malignancy risk as well as lesions with significant risk of concurrent neoplasia. Diagnosis of B3 lesions in screening populations is largely made on needle core biopsy, which provides little tissue to adequately diagnose pathologically diverse lesions. The advent of vacuum-assisted biopsy (VAB) provides the multidisciplinary team with a more representative pathology sample to direct management. Methods In this unit, in 2009, a pathway to guide management of B3 lesions detected on needle core biopsy in screening patients was implemented to assess whether VAB was a safe and viable alternative to surgery in selected cases. Here we present the 5-year follow-up results of this pathway. Results 398 patients with B3 lesions were suitable for this pathway, of which 321 went on to have second-line VAB. 24% of these patients subsequently required surgery for malignancy or ongoing concerns, and thus 245 avoided surgery being subsequently referred for 5-year mammographic surveillance or back to screening. Median follow-up was 3 years (IQR 2), and no cancers were detected at the original B3 site during follow-up. Conclusions We have demonstrated here that with large volume tissue sampling for indeterminate lesions of the breast surgery can be safely avoided in selected B3 lesions with and without atypia.

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.

PSMD9 expression predicts radiotherapy response in breast cancer

BackgroundMore than 50% of cancer patients are recommended to receive radiotherapy. Recommendations are based mainly on clinical and pathological factors and not intrinsic tumour radio-sensitivity. Use of radiotherapy according to predictive markers would potentially reduce costly over-treatment, and improve the treatment risk-benefit ratio and cancer outcomes. Tumour expression of the 26S proteasome has been reported to predict radiotherapy response: low expression was associated with higher rates of local recurrence after radiotherapy, suggesting that low proteasome expression and activity was associated with radio-resistance. However, this conclusion is at odds with the emerging use of proteasome inhibitors as radio-sensitizers. Our aim was to further analyse the relevance of 26S proteasome expression, focussing specifically on the PSMD9 subunit, in the largest clinical cohort to date, and to investigate the functional role of PSMD9 in radio-sensitivity in breast cancer cell lines.MethodsWe examined expression of PSMD9 using immunohistochemistry in a cohort of 157 breast cancer patients, including 32 cases (20.4%) that subsequently developed local recurrences. The value of expression as a prognostic or radiotherapy predictive marker was tested using Kaplan-Meier and Cox regression analyses. PSMD9 function was examined in breast cancer cell lines MCF7 and MDA-MB-231 using siRNA knock-downs and colony forming assays after irradiation.ResultsLow tumour PSMD9 expression was significantly associated with a reduced incidence of local recurrence in patients receiving adjuvant radiotherapy (univariate log rank p = 0.02; multivariate regression p = 0.009), but not in those treated without radiotherapy, suggesting that low PSMD9 expression was associated with relative tumour radio-sensitivity. In support of this, reduction of PSMD9 expression using siRNA in breast cancer cell lines in vitro sensitized cells to radiotherapy.ConclusionsWe conclude that PSMD9 expression may predict radiotherapy benefit, with low expression indicative of relative radio-sensitivity, the opposite of previous reports relating to 26S proteasome expression. Our conclusion is compatible with use of proteasome inhibitors as radio-sensitizers, and highlights PSMD9 as a potential target for radio-sensitizing drugs.

Neoadjuvant chemotherapy with MRI monitoring for breast cancer

Neoadjuvant chemotherapy (NACT) is increasingly being offered to patients with breast cancer. No survival benefit has been demonstrated for NACT, but it may serve to reduce tumour size and improve prognosis through the attainment of a pathological complete response (pCR). The role and mode of MRI monitoring during NACT remain unclear.

Digital Pathology for the Primary Diagnosis of Breast Histopathological Specimens: An Innovative Validation and Concordance Study

To train and individually validate a group of breast pathologists in specialty‐specific digital primary diagnosis by using a novel protocol endorsed by the Royal College of Pathologists’ new guideline for digital pathology. The protocol allows early exposure to live digital reporting, in a risk‐mitigated environment, and focuses on patient safety and professional development.

Expression of phosphorylated eIF4E-binding protein 1, but not of eIF4E itself, predicts survival in male breast cancer.

Background:Male breast cancer is rare and treatment is based on data from females. High expression/activity of eukaryotic initiation factor 4E (eIF4E) denotes a poor prognosis in female breast cancer, and the eIF4E pathway has been targeted therapeutically. Eukaryotic initiation factor 4E activity in female breast cancer is deregulated by eIF4E overexpression and by phosphorylation of its binding protein, 4E-BP1, which relieves inhibitory association between eIF4E and 4E-BP1. The relevance of the eIF4E pathway in male breast cancer is unknown.Methods:We have assessed expression levels of eIF4E, 4E-BP1, 4E-BP2 and phosphorylated 4E-BP1 (p4E-BP1) using immunohistochemistry in a large cohort of male breast cancers (n=337) and have examined correlations with prognostic factors and survival.Results:Neither eIF4E expression nor estimated eIF4E activity were associated with prognosis. However, a highly significant correlation was found between p4E-BP1 expression and disease-free survival (DFS), linking any detectable p4E-BP1 with poor survival (univariate log rank P=0.001; multivariate HR 8.8, P=0.0001).Conclusions:Our data provide no support for direct therapeutic targeting of eIF4E in male breast cancer, unlike in females. However, as p4E-BP1 gives powerful prognostic insights that are unrelated to eIF4E function, p4E-BP1 may identify male breast cancers potentially suitable for therapies directed at the upstream kinase, mTOR.

Is surgical diagnostic excision always necessary for solid lesions with atypia

As part of diagnostic work for radiological abnormalities seen in the breast, there has been an increase in use of vacuum-assisted biopsies for diagnosis. This allows more tissue to be sampled and therefore leads to a greater degree of diagnostic accuracy. In addition to diagnosis, in some centres the same procedure has also been used for removal of the entire lesion--vacuum-assisted excision (VAE). This is sometimes offered in place of a diagnostic surgical excision in cases of B3 lesions. We wanted to examine whether VAE can be a safe alternative for B3 lesion that show atypia.