Supplementary Materialsoncotarget-10-133-s001. and effector functions of RasGRF2 underlie at least in part, the AnxA6 mediated TNBC cell growth and/or motility, but also provide a rationale to target Ras-driven TNBC with EGFR targeted therapies in combination with inhibition of RasGRF2. = 8). EZR The growth of the xenograft AR-C69931 enzyme inhibitor tumors was monitored over time (A) and tumor size and weight (B and C) were determined following euthanasia of the tumor bearing mice. (D) Nu/J mice were injected with the indicated numbers of AnxA6-deficient BT-A6A cells and tumor volume was monitored as in (A) above. (ECF) Immunohistochemistry of xenograft tumors. (E) Formalin fixed, paraffin embedded thin sections of xenograft tumor tissues derived from AnxA6 down-regulated BT-A6sh5 and AnxA6 deficient BT-A6A cells were stained with antibodies against AnxA6, EGFR and RasGRF2 as well as with hematoxylin-eosin. (F) Immunostained tumor tissue sections were digitally scanned and quantified using the Tissue IA software (Leica Microsystems). **indicates 0.01. GCH) Intracellular Ca2+ spectrofluorimetry. Cell suspensions were loaded with fura-2 AM and changes in intracellular Ca2+ concentration were recorded in real time using the Hitachi F2500 spectrofluorimeter. Representative traces showing activation of store-operated Ca2+ influx by treatment of BT-NSC and BT-A6A cells with EGF followed by addition of Ca2+ (H) or AR-C69931 enzyme inhibitor by treatment of BT-NSC and BT-A6sh5 with ionomycin followed by addition of Ca2+ (G). Given that reduced expression of AnxA6 is associated with increased AR-C69931 enzyme inhibitor expression of the Ca2+-activated RasGRF2 (Figure ?(Figure2),2), we speculated that increased levels of RasGRF2 may drive the rapid growth of the xenograft tumors following AnxA6 down-regulation or loss in BT-549 cells. To test this, we stained the tumor tissues derived from the BT-A6sh5 cells and AnxA6-deficient BT-A6A cells by immunohistochemistry. As expected, AnxA6 was barely detected in xenograft tumors derived from AnxA6 deficient BT-A6A cells compared to that in tumors derived from BT-A6sh5 cells (Figure ?(Figure3E3E and ?and3F).3F). Consistent with AR-C69931 enzyme inhibitor our recent report , the expression of EGFR was also decreased by 2-fold in tumors derived from AnxA6 deficient cells compared to that in tumors derived from AnxA6 down-regulated BT-A6sh5 cells (Figure ?(Figure3E3E and ?and3F).3F). Surprisingly, the expression level of RasGRF2 in tumors from AnxA6-deficient cells, was 2-fold lower than that in tumors derived from AnxA6-depleted BT-A6sh5 cells (Figure ?(Figure3E3E and ?and3F).3F). Since the activity of RasGRF2 is Ca2+ dependent and activation of RasGRF2 has been shown to be accompanied by its down-regulation , we speculated that reduced expression or loss of AnxA6 may be associated with higher cytosolic Ca2+ levels and/or deregulated Ca2+ influx. To AR-C69931 enzyme inhibitor test this, we assessed the intracellular Ca2+ dynamics by spectrofluorimetry. We show that control AnxA6 expressing cells responded to EGF treatment with release of Ca2+ from intracellular stores and this was accompanied by store operated Ca2+ entry in the presence of up to 2.5 mM Ca2+. On the contrary, AnxA6 deficient BT-A6A cells apparently lost their responsiveness to EGF and showed deregulated Ca2+ entry in the presence of 2.5 mM Ca2+ and consequently higher cytosolic Ca2+ levels (Figure ?(Figure3G).3G). We next showed that following ionomycin treatment, intracellular Ca2+ levels were higher in AnxA6 depleted BT-A6sh5 cells compared to AnxA6 expressing control cells (Figure ?(Figure3H).3H). Meanwhile, modest AnxA6 down-regulation was associated with reduced responsiveness to EGF but did not significantly alter the Ca2+ influx dynamics compared to control AnxA6 expressing cells (data not shown). Together with data in Figure ?Figure2,2, these data suggest that the reciprocal expression of AnxA6 and RasGRF2 in TNBC cells is dependent at least in part, on AnxA6 regulated plasma membrane permeability to extracellular Ca2+. Up-regulation of AnxA6 is associated with increased Cdc42 activity and cell.