ECM1 secreted by HER2-overexpressing breast cancer cells promotes formation of a vascular niche accelerating cancer cell migration and invasion.

The tumor microenvironment is increasingly recognized as key player in cancer progression. Investigating heterotypic interactions between cancer cells and their microenvironment is important for understanding how specific cell types support cancer. Forming the vasculature, endothelial cells (ECs) are a prominent cell type in the microenvironment of both normal and neoplastic breast gland. Here, we sought out to analyze epithelial-endothelial cross talk in the breast using isogenic non-tumorigenic vs. tumorigenic breast epithelial cell lines and primary ECs. The cellular model used here consists of D492, a breast epithelial cell line with stem cell properties, and two isogenic D492-derived EMT cell lines, D492M and D492HER2. D492M was generated by endothelial-induced EMT and is non-tumorigenic while D492HER2 is tumorigenic, expressing the ErbB2/HER2 oncogene. To investigate cellular cross talk, we used both conditioned medium (CM) and 2D/3D co-culture systems. Secretome analysis of D492 cell lines was performed using mass spectrometry and candidate knockdown (KD), and overexpression (OE) was done using siRNA and CRISPRi/CRISPRa technology. D492HER2 directly enhances endothelial network formation and activates a molecular axis in ECs promoting D492HER2 migration and invasion, suggesting an endothelial feedback response. Secretome analysis identified extracellular matrix protein 1 (ECM1) as potential angiogenic inducer in D492HER2. Confirming its involvement, KD of ECM1 reduced the ability of D492HER2-CM to increase endothelial network formation and induce the endothelial feedback, while recombinant ECM1 (rECM1) increased both. Interestingly, NOTCH1 and NOTCH3 expression was upregulated in ECs upon treatment with D492HER2-CM or rECM1 but not by CM from D492HER2 with ECM1 KD. Blocking endothelial NOTCH signaling inhibited the increase in network formation and the ability of ECs to promote D492HER2 migration and invasion. In summary, our data demonstrate that cancer-secreted ECM1 induces a NOTCH-mediated endothelial feedback promoting cancer progression by enhancing migration and invasion. Targeting this interaction may provide a novel possibility to improve cancer treatment.

Defining new reference intervals for serum free light chains in individuals with chronic kidney disease: Results of the iStopMM study.

Serum free light chain (FLC) concentration is greatly affected by kidney function. Using a large prospective population-based cohort, we aimed to establish a reference interval for FLCs in persons with chronic kidney disease (CKD). A total of 75422 participants of the iStopMM study were screened with serum FLC, serum protein electrophoresis and immunofixation. Estimated glomerular filtration rate (eGFR) was calculated from serum creatinine. Central 99% reference intervals were determined, and 95% confidence intervals calculated. Included were 6461 (12%) participants with measured FLCs, eGFR < 60 mL/min/1.73 m2, not receiving renal replacement therapy, and without evidence of monoclonality. Using current reference intervals, 60% and 21% had kappa and lambda FLC values outside the normal range. The FLC ratio was outside standard reference interval (0.26-1.65) in 9% of participants and outside current kidney reference interval (0.37-3.10) in 0.7%. New reference intervals for FLC and FLC ratio were established. New reference intervals for the FLC ratio were 0.46-2.62, 0.48-3.38, and 0.54-3.30 for eGFR 45-59, 30-44, and < 30 mL/min/1.73 m2 groups, respectively. The crude prevalence of LC-MGUS in CKD patients was 0.5%. We conclude that current reference intervals for FLC and FLC ratio are inaccurate in CKD patients and propose new eGFR based reference intervals to be implemented.

High expression of the vacuole membrane protein 1 (VMP1) is a potential marker of poor prognosis in HER2 positive breast cancer.

BACKGROUND: Fusion genes result from genomic structural changes, which can lead to alterations in gene expression that supports tumor development. The aim of the study was to use fusion genes as a tool to identify new breast cancer (BC) genes with a role in BC progression. METHODS: Fusion genes from breast tumors and BC cell lines were collected from publications. RNA-Seq data from tumors and cell lines were retrieved from databanks and analyzed for fusions with SOAPfuse or the analysis was purchased. Fusion genes identified in both tumors (n = 1724) and cell lines (n = 45) were confirmed by qRT-PCR and sequencing. Their individual genes were ranked by selection criteria that included correlation of their mRNA level with copy number. The expression of the top ranked gene was measured by qRT-PCR in normal tissue and in breast tumors from an exploratory cohort (n = 141) and a validation cohort (n = 277). Expression levels were correlated with clinical and pathological factors as well as the patients' survival. The results were followed up in BC cohorts from TCGA (n = 818) and METABRIC (n = 2509). RESULTS: Vacuole membrane protein 1 (VMP1) was the most promising candidate based on specific selection criteria. Its expression was higher in breast tumor tissue than normal tissue (p = 1x10-4), and its expression was significantly higher in HER2 positive than HER2 negative breast tumors in all four cohorts analyzed. High expression of VMP1 associated with breast cancer specific survival (BCSS) in cohort 1 (hazard ratio (HR) = 2.31, CI 1.27-4.18) and METABRIC (HR = 1.26, CI 1.02-1.57), and also after adjusting for HER2 expression in cohort 1 (HR = 2.03, CI 1.10-3.72). BCSS was not significant in cohort 2 or TCGA cohort, which may be due to differences in treatment regimens. CONCLUSIONS: The results suggest that high VMP1 expression is a potential marker of poor prognosis in HER2 positive BC. Further studies are needed to elucidate how VMP1 could affect pathways supportive of tumorigenesis.

A genome-wide study of allelic imbalance in human testicular germ cell tumors using microsatellite markers.

Testicular germ cell tumors (TGCT) arise by multistep carcinogenesis pathways involving selective losses and gains of chromosome material. To locate cancer genes underlying this selection, we performed a genome-wide study of allelic imbalance (AI) in 32 tumors, using 710 microsatellite markers. The highest prevalence of AI was found at 12p, in line with previous studies finding consistent gain of the region in TGCTs. High frequency of AI was also observed at chromosome arms 4p, 9q, 10p, 11q, 11p, 13q, 16q, 18p, and 22q. Within 39 candidate regions identified by mapping of smallest regions of overlap (SROs), the highest frequency of AI was at 12p11.21 approximately p11.22 (62%), 12p12.1 approximately p13.1 (53%), 12p13.1 approximately p13.2 (53%), 11q14.1 approximately q14.2 (53%), 11p13 approximately p14.3 (47%), 9q21.13 approximately q21.32 (47%), and 4p15.1 approximately p15.2 (44%). Two genes known to be involved in cancer reside in these regions, ETV6 at 12p13.2 (TEL oncogene) and WT1 at 11p13. We also found a significant association (P = 0.02) between AI at 10q21.1 approximately q22.2 and higher clinical stage. This study contributes to the ongoing search for genes involved in transformation of germ cells and provides a useful reference point to previous studies using cytogenetic techniques to map chromosome changes in TGCTs.