Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B , BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug development.

Insulin analogues are widely used in clinical practice. Modifications on the insulin molecular structure can affect the affinity and activation towards two closely related receptor tyrosine kinases: the insulin receptor (INSR) and the insulin-like growth factor 1 receptor (IGF1R). A switch towards higher IGF1R affinity is likely to emphasize mitogenesis rather than glucose metabolism. Relevant well-validated experimental tools to address the insulin analogue activation of either INSR or IGF1R are missing. We have established a panel of human MCF-7 breast cancer cell lines either ectopically expressing the INSR (A or B isoform) in conjunction with a stable knockdown of the IGF1R or ectopically expressing the IGF1R in conjunction with a stable knockdown of the INSR. In these cell lines, we systematically evaluated the INSR and IGF1R receptor activation and downstream mitogenic signalling of all major clinical relevant insulin analogues in comparison with insulin and IGF1R. While most insulin analogues primarily activated the INSR, the mitogenic activation pattern of glargine was highly similar to IGF1 and insulin AspB10, known to bind IGF1R and induce carcinogenesis. Yet, in a long-term proliferation assay, the proliferative effect of glargine was not much different from regular insulin or other insulin analogues. This was caused by the rapid enzymatic conversion into its two metabolic active metabolites M1 and M2, with reduced mitogenic signalling through the IGF1R. In summary, based on our new cell models, we identified a similar mitogenic potency of insulin glargine and AspB10. However, rapid enzymatic conversion of glargine precludes a sustained activation of the IGF1R signalling pathway.

The efficacy of prospective cancer treatments is routinely estimated by in vitro cell-line proliferation screens. However, it is unclear whether tumor aggressiveness and patient survival are influenced more by the proliferative or the migratory properties of cancer cells. To address this question, we experimentally measured proliferation and migration phenotypes across more than 40 breast cancer cell-lines. Based on the latter, we built and validated individual predictors of breast cancer proliferation and migration levels from the cells’ transcriptomics. We then apply these predictors to estimate the proliferation and migration levels of more than 1000 TCGA breast cancer tumors. Reassuringly, both estimates increase with tumor’s aggressiveness, as qualified by its stage, grade, and subtype. However, predicted tumor migration levels are significantly more strongly associated with patient survival than the proliferation levels. We confirmed these findings by conducting siRNA knock-down experiments on the highly migratory MDA-MB-231 cell lines and deriving gene knock-down based proliferation and migration signatures. We show that cytoskeletal drugs might be more beneficial in patients with high predicted migration levels. Taken together, these results testify to the importance of migration levels in determining patient survival.

IntroductionInsulin analogues are structurally modified molecules with altered pharmaco-kinetic and -dynamic properties compared to regular human insulin used by diabetic patients. While these compounds are tested for undesired mitogenic effects, an epidemiological discussion is ongoing regarding an association between insulin analogue therapy and increased cancer incidence, including breast cancer. Standard in vivo rodent carcinogenesis assays do not pick up this possible increased carcinogenic potential.MethodsHere we studied the role of insulin analogues in breast cancer development. For this we used the human relevant mammary gland specific p53R270H/+WAPCre mouse model. Animals received life long repeated treatment with four different insulin (−like) molecules: normal insulin, insulin glargine, insulin X10 (AspB10) or insulin-like growth factor 1 (IGF1).ResultsInsulin-like molecules with strong mitogenic signaling, insulin X10 and IGF1, significantly decreased the time for tumor development. Yet, insulin glargine and normal insulin, did not significantly decrease the latency time for (mammary gland) tumor development. The majority of tumors had an epithelial to mesenchymal transition phenotype (EMT), irrespective of treatment condition. Enhanced extracellular signaling related kinase (Erk) or serine/threonine kinase (Akt) mitogenic signaling was in particular present in tumors from the insulin X10 and IGF1 treatment groups.ConclusionsThese data indicate that insulin-like molecules with enhanced mitogenic signaling increase the risk of breast cancer development. Moreover, the use of a tissue specific cancer model, like the p53R270H/+WAPCre mouse model, is relevant to assess the intrinsic pro-carcinogenic potential of mitogenic and non-mitogenic biologicals such as insulin analogues.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0518-y) contains supplementary material, which is available to authorized users.