Another study Tofacitinib alopecia demonstrated that activated PKC induces lamellipodia formation and subsequent increased migratory activity of subconjunctival fibroblasts [19]. Thus, it has been suggested that PKCs induce cell proliferation/survival, invasion and metastasis. In this study, ANX7 was identified as a novel binding partner of BART, and BART was found to be associated with ANX7-PKC complex formation in PDAC cells. Furthermore, the current results demonstrated that knocking down BART induces cell invasion by increasing PKC�� activity through the loss of ANX7-PKC�� complex formation. Thus, decreased active PKC�� via both BART and ANX7 contributes to inhibition of PDAC invasiveness. Results BART binds to ANX7 in PDAC cells BART knockdown increases retroperitoneal invasion and PDAC cell metastasis to liver in an orthotopic xenograft model, as described in a previous report [4].
To investigate the mechanism by which BART suppresses invasiveness and metastasis, immunoprecipitation (IP) experiments were performed in the human PDAC cell line S2-013 using a specific antibody to BART, to detect complexes of BART with other proteins. S2-013 is a cloned subline of a PDAC cell line (SUIT-2) derived from a liver metastasis [20], and was obtained from Dr. T. Iwamura (Miyazaki Medical College, Miyazaki, Japan). Silver-stained immunoprecipitated fractions separated on SDS-PAGE gels revealed a 50-kDa band that was not seen in the isotype control immunoprecipitates (arrow in Fig. 1A). The band was excised and analyzed by Q-TOF-MS after in-gel trypsin digestion, and identified as ANX7.
The peptide sequence coverage was 15% (Fig. 1B). This specific binding of ANX7 to BART was demonstrated by co-IP from S2-013 cells (Fig. 1C) and subcellular colocalization was analyzed by immunostaining of S2-013 cells (Fig. 1D). BART and ANX7 coimmunoprecipitated and were colocalized in the cytoplasm. Of note is that BART and ANX7 accumulated in lamellipodial-like protrusions that are essential for cell migration (arrows in Fig. 1E). Figure 1 BART binds to ANX7 in lamellipodial-like protrusions. ANX7 inhibits PDAC cell invasion Previously, cell clones were generated in which BART was stably suppressed by vector-based specific short hairpin small interfering RNA (siRNA) in S2-013 cells that formerly expressed high levels of BART [4]. To determine the function of BART-ANX7 complexes, a wound-healing Dacomitinib immunostaining assay was used to observe the localization of BART and ANX7 in polarized migrating cells (Fig. 2A). Both BART and ANX7 were recruited to the leading edges during wound healing of control S2-013 cells (arrows in Fig. 2A). Depletion of BART inhibited ANX7 accumulation at the leading edges (lower panels in Fig. 2A). Combined with the result of Fig.