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Accumulating evidence from clinical trials indicates that specific targeting of the IGF1 receptor (IGF1R) is not efficient as an anti-breast cancer treatment. One possible reason is that the mitogenic signals from the insulin receptor (IR) can be processed independently or as compensation to inhibition of the IGF1R. In this study, we highlight the role of the IR in mediating breast tumor progression in both WT mice and a hyperinsulinemic MKR mouse model by induction of Ir (Insr) or Igf1r knockdown (KD) in the mammary carcinoma Mvt-1 cell line. By using the specific IR antagonist-S961, we demonstrated that Igf1r-KD induces elevated responses by the IR to IGF1. On the other hand, Ir-KD cells generated significantly smaller tumors in the mammary fat pads of both WT and MKR mice, as opposed to control cells, where as the Igf1r-KD cells did not. The tumorigenic effects of insulin on the Mvt-1 cells were also demonstrated using microarray analysis, which indicates alteration of genes and signaling pathways involved in proliferation, the cell cycle, and apoptosis following insulin stimulation. In addition, the correlation between IR and the potential prognostic marker for aggressive breast cancer, CD24, was examined in the Ir-KD cells. Fluorescence-activated cell sorting (FACS) analysis revealed more than 60% reduction in CD24 expression in the Ir-KD cells when compared with the control cells. Our results also indicate that CD24-expressing cells can restore, at least in part, the tumorigenic capacity of Ir-KD cells. Taken together, our results highlight the mitogenic role of the IR in mammary tumor progression with a direct link to CD24 expression.

IntroductionBreast tumors are comprised of distinct cancer cell populations which differ in their tumorigenic and metastatic capacity. Characterization of cell surface markers enables investigators to distinguish between cancer stem cells and their counterparts. CD24 is a well-known cell surface marker for mammary epithelial cells isolation, recently it was suggested as a potential prognostic marker in a wide variety of malignancies. Here, we demonstrate that CD24+ cells create intra-tumor heterogeneity, and display highly metastatic properties.MethodsThe mammary carcinoma Mvt1 cells were sorted into CD24− and CD24+ cells. Both subsets were morphologically and phenotypically characterized, and tumorigenic capacity was assessed via orthotopic inoculation of each subset into the mammary fat pad of wild-type and MKR mice. The metastatic capacity of each subset was determined with the tail vein metastasis assay. The role of CD24 in tumorigenesis was further examined with shRNA technology. GFP-labeled cells were monitored in vivo for differentiation. The genetic profile of each subset was analyzed using RNA sequencing.ResultsCD24+ cells displayed a more spindle-like cytoplasm. The cells formed mammospheres in high efficiency and CD24+ tumors displayed rapid growth in both WT and MKR mice, and were more metastatic than CD24- cells. Interestingly, CD24-KD in CD24+ cells had no effect both in vitro and in vivo on the various parameters studied. Moreover, CD24+ cells gave rise in vivo to the CD24− that comprised the bulk of the tumor. RNA-seq analysis revealed enrichment of genes and pathways of the extracellular matrix in the CD24+ cells.ConclusionCD24+ cells account for heterogeneity in mammary tumors. CD24 expression at early stages of the cancer process is an indication of a highly invasive tumor. However, CD24 is not a suitable therapeutic target; instead we suggest here new potential targets accounting for early differentiated cancer cells tumorigenic capacity.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0589-9) contains supplementary material, which is available to authorized users.

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One dose of metyrapone (1.5g) administered at 06.00 h, with subsequent measurement of 1 1 -d e o x ycortisol and 17-hydroxycort i cost eroi d (17-OHCS) levels in plasma at 12.00 and 14.00 h, allowed accurate assessment of the pituitary-adrenal reserve. Normal response was defined as achieving a serum 17-OHCS level of more than 10.0 \g=m\g/100 ml and a 11-deoxycortisol level of more than 6.0 \g=m\g/100 ml at either 12.00 or 14.00 h.These criteria are based on a group of 18 persons with normal pituitary-adrenal axis, and 86 additional cases responded in this normal range. In this group of 104 subjects, 11-deoxycortisol levels rose to 9.2 \ m=+-\ 3.5 \ g=m\ g/ 100 ml at noon and 17-OHCS levels to 15.4 \ m=+-\ 4.7 \g=m\g/100 ml at 14.00 h. Post-metyrapone 17-OHCS levels were significantly higher than normal cortisol levels at these times (P < 0.001) and than those observed at 08.00 h on the day of the test, demonstrating stimulation of adrenal corticoid production in addition to blockade of cortisol production by metyrapone. Thi rty\x=req-\ one patients found to suffer from secondary adrenal failure showed impaired response. All these patients had limited pituitary-adrenal reserve, either proven by other pituitary-adrenal tests or implicated by severe pituitary disease.The metyrapone test was introduced by Liddle et al. (1959) and has since been widely accepted as an accurate means of assessing pituitary-adrenal reserve. By blocking 11-ß-hydroxylation of steAddress requests for reprints to: