Sperm Flagellar 1 Binds Actin in Intestinal Epithelial Cells and Contributes to Formation of Filopodia and Lamellipodia.

BACKGROUND & AIMS: Sperm flagellar 1 (also called CLAMP) is a microtubule-associated protein that regulates microtubule dynamics and planar cell polarity in multi-ciliated cells. We investigated the localization and function of sperm flagellar 1, or CLAMP, in human intestinal epithelia cells (IECs). METHODS: We performed studies with SKCO-15 and human intestinal enteroids established from biopsies from different intestinal segments (duodenal, jejunum, ileal, and colon) of a single donor. Enteroids were induced to differentiation after incubation with growth factors. The distribution of endogenous CLAMP in IECs was analyzed by immunofluorescence microscopy using total internal reflection fluorescence-ground state depletion and confocal microscopy. CLAMP localization was followed during the course of intestinal epithelial cell polarization as cells progressed from flat to compact, confluent monolayers. Protein interactions with endogenous CLAMP were determined in SKCO-15 cells using proximity ligation assays and co-immunoprecipitation. CLAMP was knocked down in SKCO-15 monolayers using small hairpin RNAs and cells were analyzed by immunoblot and immunofluorescence microscopy. The impact of CLAMP knock-down in migrating SKCO-15 cells was assessed using scratch-wound assays. RESULTS: CLAMP bound to actin and apical junctional complex proteins but not microtubules in IECs. In silico analysis predicted the calponin-homology domain of CLAMP to contain conserved amino acids required for actin binding. During IEC polarization, CLAMP distribution changed from primarily basal stress fibers and cytoplasm in undifferentiated cells to apical membranes and microvilli in differentiated monolayers. CLAMP accumulated in lamellipodia and filopodia at the leading edge of migrating cells in association with actin. CLAMP knock-down reduced the number of filopodia, perturbed filopodia polarity, and altered the organization of actin filaments within lamellipodia. CONCLUSIONS: CLAMP is an actin-binding protein, rather than a microtubule-binding protein, in IECs. CLAMP distribution changes during intestinal epithelial cell polarization, regulates the formation of filopodia, and appears to assist in the organization of actin bundles within lamellipodia of migrating IECs. Studies are needed to define the CLAMP domains that interact with actin and whether its loss from IECs affects intestinal function.

IL-1ß Inflammatory Cytokine-Induced TP63 Isoform ∆NP63α Signaling Cascade Contributes to Cisplatin Resistance in Human Breast Cancer Cells.

The mechanisms behind the induction of malignancy and chemoresistance in breast cancer cells are still not completely understood. Inflammation is associated with the induction of malignancy in different types of cancer and is highlighted as an important factor for chemoresistance. In previous work, we demonstrated that the inflammatory cytokine interleukin 1ß (IL-1ß)-induced upregulation of genes was associated with chemoresistance in breast cancer cells. Here, we evaluated the participation and the expression profile of TP63 in the induction of resistance to cisplatin. By loss-of-function assays, we identified that IL-1ß particularly upregulates the expression of the tumor protein 63 (TP63) isoform ΔNP63α, through the activation of the IL-1ß/IL-1RI/ß-catenin signaling pathway. Upregulation of ΔNP63α leads to an increase in the expression of the cell survival factors epidermal growth factor receptor (EGFR) and phosphatase 1D (Wip1), and a decrease in the DNA damage sensor, ataxia-telangiectasia mutated (ATM). The participation of these processes in the increase of resistance to cisplatin was confirmed by silencing TP63 expression or by inhibition of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) activity in the IL-1ß/IL-1RI/ß-catenin signaling pathway. These data reinforced the importance of an inflammatory environment in the induction of drug resistance in cancer cells and uncovered a molecular mechanism where the IL-1ß signaling pathway potentiates the acquisition of cisplatin resistance in breast cancer cells.

IL-1ß induced methylation of the estrogen receptor ERα gene correlates with EMT and chemoresistance in breast cancer cells.

Inflammation has been recently acknowledged as a key participant in the physiopathology of oncogenesis and tumor progression. The inflammatory cytokine IL-1ß has been reported to induce the expression of markers associated with malignancy in breast cancerous cells through Epithelial-Mesenchymal Transition (EMT). Aggressive breast cancer tumors classified as Triple Negative do not respond to hormonal treatment because they lack three crucial receptors, one of which is the estrogen receptor alpha (ERα). Expression of ERα is then considered a good prognostic marker for tamoxifen treatment of this type of cancer, as the binding of this drug to the receptor blocks the transcriptional activity of the latter. Although it has been suggested that inflammatory cytokines in the tumor microenvironment could regulate ERα expression, the mechanism(s) involved in this process have not yet been established. We show here that, in a cell model of breast cancer cells (6D cells), in which the inflammatory cytokine IL-1ß induces EMT by activation of the IL-1ß/IL-1RI/ß-catenin pathway, the up regulation of TWIST1 leads to methylation of the ESR1 gene promoter. This epigenetic modification produced significant decrease of the ERα receptor levels and increased resistance to tamoxifen. The direct participation of IL-1ß in these processes was validated by blockage of the cytokine-induced signaling pathway by wortmannin inactivation of the effectors PI3K/AKT. These results support our previous reports that have suggested direct participation of the inflammatory cytokine IL-1ß in the transition to malignancy of breast cancer cells.