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  • Redundancy of tumor promoting signaling pathways is


    Redundancy of tumor promoting signaling pathways is one of the mechanisms that prevent a lasting effect of targeted therapies, as has been demonstrated by the growing number of alternative pathways that confer resistance to EGFR-targeted therapies [3]. We show here that CSF-1R can, at least partially, compensate for gefitinib-suppressed EGFR signaling in A431 cells. Our work is in line with indications from other cell systems that CSF-1R and EGFR execute a similar function in epithelial cells. First, invasiveness of MDA-MB-231 is driven by an autocrine CSF-1/CSF-1R loop as well as by a paracrine loop involving EGF produced by tumor-associated macrophages [18]. Second, non-transformed mammary cell line MCF-10A became independent of EGF for growth and survival by overexpression of CSF-1R and stimulation with CSF-1 [51]. Third, a partial redundancy of CSF-1R and EGFR signaling has been postulated by phosphotyrosine proteome analysis of CSF-1R overexpressing MCF-10A Dihydrotestosterone and comparison with EGFR signaling in other cell systems [52]. What are the cellular requirements allowing CSF-1R to reduce sensitivity to gefitinib? Our data suggested that PI3K/Akt, Mek/Erk1/2, PKCs and STAT3 Dihydrotestosterone were involved in CSF-1R-mediated proliferation of gefitinib treated A431 cells, but not STAT5. It has been reported that CSF-1R induced src-dependent phosphorylation of STAT5 in MCF-10A cells [52]. We did not observe activation of members of the src family (data not shown). Morandi et al. [23] suggested that CSF-1R might support proliferation in breast cancer cell line SK-BR-3 based on examination of DNA-synthesis and expression of proliferation-related genes. However, a direct demonstration that CSF-1R activity increases the number of cells is missing. When we treated SK-BR-3 cells with gefitinib, CSF-1 failed to rescue cell proliferation. However, CSF-1 robustly induced phosphorylation of Erk1/2 and Akt but not STAT3 (data not shown). These data indicate that the exact cellular context regarding oncogenic alterations and/or other cell type specific properties that allow CSF-1R to induce proliferation in epithelial cancer cells remain to be defined in more detail, and further studies will have to investigate clinical relevance of CSF-1R expression regarding targeted therapies. However, using publicly available RNA-seq data we demonstrated that CSF1R mRNA is expressed in a number of carcinoma cell lines of different tumor types, including tumors that are very aggressive and difficult to treat such as hepatocellular carcinoma or lung cancer. Expression of CSF-1R is normally restricted to monocytes/macrophages, osteoclasts, and a few non-hematopoietic cell types including epithelial intestinal cells of the colon and certain subpopulations of neurons [36]. In addition, CSF-1R is transiently expressed during processes that require tissue remodeling. Examples include pregnancy with expression of CSF-1R in trophoblast and uterine epithelium, the mammary ductal network of the breast epithelium during lactation [36, 53], or renal tubular epithelium after injury [54]. A fraction of solid tumor cells with aberrant expression of CSF-1R may be related to these cell types. However, cells undergo extensive changes in chromatin structure, transcription and translation during tumorigenesis. Interestingly, human telomerase reverse transcriptase (hTERT)-mediated immortalization upregulates expression of CSF1R in ovarian surface epithelial cells [55]. However, the molecular mechanism that allows aberrant transcription of CSF1R in solid cancer is unclear. It has been demonstrated that expression of CSF1R can be induced by transforming growth factor (TGF) beta1 [5, 17, 18]. Moreover, CSF1R expression can be hormonally regulated via a glucocorticoid response element in the trophoblast-specific promotor in breast and cervical cancer [5, 11, [56], [57], [58]]. Of note, the expression of CSF1R in humans is driven by a monocyte-macrophage-specific promotor immediately upstream of exon 2 or a trophoblast-specific promotor upstream of non-coding exon 1 that is not conserved in rodents [59]. Furthermore, Lamprecht et al. [60] have demonstrated aberrant expression of CSF1R in certain lymphomas driven by a long terminal repeat (LTR) sequence in intron 1. By closer inspection of RNA-seq raw data (from [26]) of 19 carcinoma cell lines, we noticed indications for expression from the LTR [60] and/or the trophoblast-specific promotor. Furthermore, as shown for MDA-MB-231 cells (Fig. 2A and Supplementary Fig. 1A), 6 cell lines expressed exons 11 to 22 stronger than exons 1 to 10. However, xenograft experiments using MDA-MB-231 cells suggest that expression of CSF1R may change under the influence of tumor microenvironment [18]. It is obvious that our analysis of cell lines that were cultured under standard conditions did not take inducible upregulation into account. Future studies on single cell level in primary tumor material may, therefore, uncover a CSF-1R expression pattern distinct from cell lines. However, inducible expression may occur only transiently and/or locally and may, thus, escape detection. Nevertheless, regarding the fact that CSF-1 is ubiquitously present and elevated in serum of breast, ovarian, colorectal and pancreatic tumor patients [[36], [37], [38], [39]], even local and/or transient expression of its receptor may be sufficient to allow single cancer cells to survive under targeted therapies until the cells acquire additional alterations that then provide stable resistance.