BRAF status modulates Interelukin-8 expression through a CHOP-dependent mechanism in colorectal cancer

Abstract

Inflammation might substantially contribute to the limited therapeutic success of current systemic therapies in colorectal cancer (CRC). Amongst cytokines involved in CRC biology, the proinflammatory chemokine IL-8 has recently emerged as a potential prognostic/predictive biomarker. Here, we show that BRAF mutations and PTEN-loss are associated with high IL-8 levels in CRC models in vitro and that BRAF/MEK/ERK, but not PI3K/mTOR, targeting controls its production in different genetic contexts. In particular, we identified a BRAF/ERK2/CHOP axis affecting IL-8 transcription, through regulation of CHOP subcellular localization, and response to targeted inhibitors. Moreover, RNA Pol II and an open chromatin status in the CHOP-binding region of the IL-8 gene promoter cooperate towards increased IL-8 expression, after a selective BRAF inhibition. Overall, our data show that IL-8 production is finely and differentially regulated depending on the tumor genetic context and might be targeted for therapeutic purposes in molecularly defined subgroups of CRC patients.

Fibroblast-Induced Paradoxical PI3K Pathway Activation in PTEN-Competent Colorectal Cancer: Implications for Therapeutic PI3K/mTOR Inhibition

“…com) (9). Isogenic cell lines HCT116, HK2-6, and HKE-3 were performed by Shirasawa's group by KRAS specific targeting technique (10,11). LS180, HT29, RKO, and SW480 cell lines were kindly provided by Dr. Federica Di Nicolantonio as previously described (11).…”

“…Isogenic cell lines HCT116, HK2-6, and HKE-3 were performed by Shirasawa's group by KRAS specific targeting technique (10,11). LS180, HT29, RKO, and SW480 cell lines were kindly provided by Dr. Federica Di Nicolantonio as previously described (11). Human foreskin fibroblasts (HFF), green fluorescent protein (GFP)-labeled fibroblasts, and BJ cell lines were kindly provided by Dr. Maurizio Fanciulli.…”

“…Based on previous experience of our group (11), HFF-CM was obtained under standardized culture conditions: 1x10 6 cells were seeded and after 24 h, cells were washed with PBS 1X and medium was replaced by serum-free medium (wo FBS); CM was collected after 72 h. Serum-free conditions were chosen based on the notion that production of soluble factors is tightly dependent on the type of growth media used; moreover, serum-free conditions are also necessary to characterize the pattern of expression of soluble factors released by fibroblasts (see below, paragraph 2.10), using specific ELISA and membrane-based arrays, since FBS contains basal levels of cyto/chemokines that could interfere with the detection of mediator(s) involved in the observed CM effects (12,13).…”

“…Whole cell extracts were obtained by SDS lysis buffer, containing 20 mM TrisHCl (pH 7.4) and 2% SDS, with 1X phosphatase and protease inhibitors (Thermo Fisher Scientific, Rochester, NY, USA). Protein quantification, separation, and detection were assessed as previously described (11). Membranes were probed with the primary and secondary antibodies reported in Supplementary Materials.…”

“…After 24 h from treatments, cells were fixed in 4% formaldehyde in 1X PBS for 10 min, permeabilized in 1X PBS containing 0.5% Triton X-100 for 5 min at room temperature. Coverslips were incubated with primary and secondary antibodies as previously described (11). The used antibodies are reported in Supplementary Materials.…”