Figure 4.
GKAP Regulates Cancer Cell Invasion through NMDAR Activity and Downstream Effectors FMRP and HSF1
(A) Western blot for GKAP and p-GluN2b in control and GKAP-knockdown βTC-3 cells under unstimulated cell culture conditions. The numbers below indicate levels of p-GluN2b and GKAP normalized to untransfected βTC-3.
(B) Fluorescence reporter assay was performed in βTC-3 small hairpin RNA (shRNA) control and GKAP-knockdown (KD) cells lines, comparing calcium transients induced by puffing an NMDA solution onto the cells. Clusters of cultured control βTC-3 cells or GKAP-KD βTC-3 cells were analyzed under a bright-field microscope after an NMDA solution (1 mM, 1 s) was puffed through perfusion pipette at left (upper panels). The arrows indicate the direction of puffing. βTC-3 shRNA control KD cells: 78 of 284 cells examined showed a response (orange bars). βTC-3 shRNA-GKAP KD cells: 5 of 178 cells showed minor NMDA responses (light blue bars). p < 10−11, Wilcoxon rank-sum test. Histogram of transient amplitude, denoting time-resolved fluorescence signals (sampling frequency/frame rate = 12.5 Hz). Light orange bar and blue bar indicate cells with no response.
(C) Invasion assay in control and GKAP-KD βTC-3 cells. Two-way ANOVA, Bonferroni multiple comparisons test: ∗∗p < 0.01; n.s., not significant (mean ± SEM, n = 3 invasion assay devices/condition in one experiment; two independent experiments were performed with consistent results).
(D) IHC staining of FMRP in B6 PanNETs. Similarly sized invasive versus non-invasive primary tumors on the same section were used for comparison (tumor borders marked by yellow dashed line in the representative images). Rare, multiple metastatic lesions in the liver from one mouse (indicated by the red arrowheads, tumor borders marked by yellow dashed line). Images shown are representative of an analysis of >50 PanNETs from >10 B6 RIP1Tag2 mice, one section per mouse, and all staining was performed in the same experiment. Magnified lesion is representative of >100 metastases from one liver.
(E) Western blot shows the efficiency of FMRP knockdown in βTC-3 cells. The numbers below indicate levels of FMRP normalized to GAPDH. Bar graph: invasion assay. Unpaired t test: ∗p < 0.05; ∗∗p < 0.01. FMRP #1 and #2 indicate two different siRNA constructs used (mean ± SD, n = 3 invasion assay devices per condition in one experiment; two independent experiments).
(F) Western blots comparing FMRP and p-HSF1 expression in βTC-3 cancer cells infected with control shRNA or shRNA-GKAP lentiviral vectors (left), and comparing expression of p-GluN2b, FMRP, and p-HSF1 in βTC-3 cancer cells treated with either vehicle or MK801 (right). GAPDH was used as a loading control and the numbers below indicate levels of p-GluN2b, FMRP, and p-HSF1 normalized to GAPDH. (n = 3).
(G) Left: tissue immunostaining shows expression of total and active GluN2b, FMRP, and total and active HSF1 in tumors from saline- and MK801-treated B6 RIP1Tag2 mice. Data shown are representative of 9–21 random pictures from >15 PanNETs from three mice per group. Scale bar in the blow-up picture represents 25 μm. Right: quantification of FMRP and pHSF1 expression in saline- and MK801-treated tumors. Mean ± SEM. FMRP, n = 9 pictures in saline treated groups, n = 21 in MK801-treated group; p-HSF1, n = 15 pictures in saline treated groups, n = 20 in MK801-treated group. Mann-Whitney test: ∗p < 0.05; ∗∗p < 0.01.
See also Figure S2.