Crizotinib induces Par-4 secretion from normal cells and GRP78 expression on the cancer cell surface for selective tumor growth inhibition

Abstract

Lung cancer is the leading cause of cancer-related deaths. Lung cancer cells develop resistance to apoptosis by suppressing the secretion of the tumor suppressor Par-4 protein (also known as PAWR) and/or down-modulating the Par-4 receptor GRP78 on the cell surface (csGRP78). We sought to identify FDA-approved drugs that elevate csGRP78 on the surface of lung cancer cells and induce Par-4 secretion from the cancer cells and/or normal cells in order to inhibit cancer growth in an autocrine or paracrine manner. In an unbiased screen, we identified crizotinib (CZT), an inhibitor of activated ALK/MET/ROS1 receptor tyrosine kinase, as an inducer of csGRP78 expression in ALK-negative, KRAS or EGFR mutant lung cancer cells. Elevation of csGRP78 in the lung cancer cells was dependent on activation of the non-receptor tyrosine kinase SRC by CZT. Inhibition of SRC activation in the cancer cells prevented csGRP78 translocation but promoted Par-4 secretion by CZT, implying that activated SRC prevented Par-4 secretion. In normal cells, CZT did not activate SRC and csGRP78 elevation but induced Par-4 secretion. Consequently, CZT induced Par-4 secretion from normal cells and elevated csGRP78 in the ALK-negative tumor cells to cause paracrine apoptosis in cancer cell cultures and growth inhibition of tumor xenografts in mice. Thus, CZT induces differential activation of SRC in normal and cancer cells to trigger the pro-apoptotic Par-4-GRP78 axis. As csGRP78 is a targetable receptor, CZT can be repurposed to elevate csGRP78 for inhibition of ALK-negative lung tumors.

Keywords: Par-4; SRC; apoptosis; crizotinib; csGRP78; lung cancer.

Figure 1

CZT induces csGRP78 in cancer cells by SRC kinase-dependent mechanism. A. CZT induces csGRP78 in cancer cells but not in normal cells. Lung cancer KP7B and A549 cells expressing mutant-Ras, and normal/immortalized lung epithelial BEAS-2B or fibroblasts HEL cells were treated with the indicated concentrations of CZT or vehicle. Cells were analyzed for csGRP78 as indicated in Methods. B. CZT but not the Met-inhibitor savolitinib (SAV) induces csGRP78 in cancer cells. Lung cancer cells with mutant-Ras (H1299 and H460) or mutant-EGFR (H1650 and H1975) were treated with 500 nM CZT, SAV, or vehicle (V). C. CZT induces phosphorylation of SRC in cancer cells, but not in normal/immortalized cells. Lung cancer cells (LLC-1, H1650, H1975, A549, A549TR, H1299) or normal/immortalized lung cells (BEAS-2B, HEL) were treated with the indicated concentrations of CZT or vehicle (v) for 24 h. Whole-cell lysates were subjected to western blot analysis for pY419-SRC (pSRC), total SRC, and GAPDH or actin as loading control. Fold change in pSRC and total SRC levels in response to CZT relative to vehicle is shown. D. SU6656 inhibits GRP78 transport to the cell surface. A549, H1650 or H1975 cells were treated with 1 µM CZT or vehicle in the presence or absence of SRC-inhibitor SU6656 (5 µM) for 24 h. E. Knockdown of SRC inhibits cell surface transport of GRP78. A549 or H1975 cells were treated with SRC-siRNA or control (CON)-siRNA for 24 h and then treated with 500 nM CZT or vehicle for 24 h (Left Panels). Knockdown of SRC was confirmed by western blot analysis (Right Panels). A, B, D and E. Percentage of csGRP78-positive cells was calculated and Mean + SD values are shown. *P < 0.001 by Student’s t test.

Figure 2

CZT binds to SRC. A. CZT binding (dashed circle) to SRC. CZT binds near the C-terminal domain of SRC and may disrupt the inactive state by preventing phosphorylation on the nearby tyrosine. Key interactions among CZT and SRC residues are shown. B. Pull-down experiments indicate that CZT binds to SRC but not to CSK. Whole-cell lysates of A549 cells were incubated with vehicle, biotinylated-CZT or biotinylated-ebastine (EBS), as a negative control, and streptavidin beads. Bound proteins were eluted from the beads with D-biotin. Eluates were subjected to western bot analysis with SRC antibody (Top Panel), or with CSK and GAPDH control antibody (Bottom Panel).

Figure 3

CZT induces Par-4 secretion in normal/immortalized cells. A. CZT induces Par-4 secretion in normal/immortalized cells (MEFs, HEL, BEAS-2B) and mice but not in lung cancer cells. Cells were treated with vehicle (V) or CZT (at indicated nM concentrations) for 24 h. C57BL/6 mice were treated with V or CZT for 5 days. Aliquots of the conditioned medium (CM) from the cells or mouse plasma samples were then subjected to western blot analysis for Par-4. The corresponding gels were stained with Coomassie blue, and the intensity of Par-4 bands were normalized relative to albumin. Fold change in Par-4 in response to CZT relative to vehicle is shown. B. CZT induces Par-4 secretion in normal/immortalized cells, but several other drugs do not. HEL cells were treated with vehicle or the indicated drugs (at 500 nM) for 24 h. Aliquots of the CM from the cells were then subjected to western blot analysis for Par-4. The corresponding gels were stained with Coomassie blue, and the intensity of Par-4 bands were normalized relative to albumin. Fold change in Par-4 in response to each drug relative to vehicle is shown.

Figure 4

Par-4 secreted by normal cells treated with CZT inhibits cancer cell viability and tumor growth. (A) CZT induces Par-4 secretion in mouse embryonic fibroblasts (MEFs) from Par-4^+/+ mice but not from Par-4^-/- or p53^-/- mice. The indicated MEFs were treated with CZT (1 µM) or vehicle (V) for 24 h, and the CM was examined for Par-4 secretion by western blot analysis. (B) CZT by itself does not inhibit cell viability of lung cancer cells. Lung cancer cells were treated with CZT (1 µM) or vehicle (V) for 24 h, and cell viability was examined by resazurin assays. (C) CM from CZT treated Par-4^+/+ MEFs, but not Par-4^-/- MEFs, inhibits cancer cell viability. Lung cancer cells A549, H1650, H1975, and KP7B were pretreated with CZT or vehicle (V) for 24 h and washed three times. The cells were then treated with the CM from normal cells Par-4^+/+ or Par-4^-/- MEFs treated with CZT. Prior to treating the cancer cells with the CM, we pre-incubated the CM for 30 min with Par-4 antibody, GRP78 antibody or control IgG. After 24 h of treatment, the cells were subjected to resazurin assays. Mean + SD values are shown. *P < 0.001 by Student’s t test. (D) CZT treatment inhibits the growth of xenografts derived from lung tumor cells. NSG mice were injected s.c. with H1650 or H1975 cells. When the tumors grew to 30 mm³, the mice were administered CZT (25 mg/kg body weight) or vehicle (n=5 per group) once every day for 19 consecutive days by oral gavage. (E) Inhibition of lung cancer growth by CZT in mice is Par-4-dependent. C57BL/6 mice that were either Par-4^+/+ (wild type) or Par-4^-/- were injected s.c. with KP7B cells, and the tumors were examined for response to CZT or vehicle. (D, E) Tumor volumes were determined at the indicated time intervals. Mean + SD is shown. **P < 0.01 by ANOVA test for tumor volumes at corresponding days in the vehicle control and CZT treatment groups of mice. Plasma samples from the mice treated with vehicle (V) or CZT collected at the end of the experiments were subjected to western blot analysis for Par-4 or Coomassie blue staining for albumin.

Figure 5

Model for differential effects of CZT in normal and cancer cells. CZT activates SRC in cancer cells but not normal cells by directly interacting with it, and activated SRC is required for translocation of GRP78 from the ER to the cancer cell surface. On the other hand, induction of Par-4 secretion in normal cells but not in cancer cells by CZT results in paracrine apoptosis and growth inhibition of cancer cells in co-culture studies and mouse xenograft models. As CZT does not activate SRC in normal cells, csGRP78 is not elevated and normal cells are resistant to apoptosis and growth inhibition by secreted Par-4.