Xiuhai Ren

c-Abl mediates endothelial apoptosis induced by inhibition of integrins αvβ3 and αvβ5 and by disruption of actin

Inhibition of integrins alphavbeta3 and alphavbeta5 in human brain microvascular endothelial cells (HBMECs) by the function-blocking peptide RGDfV induces loss of spreading on vitronectin, cell detachment, and apoptosis. We demonstrate that cell detachment is not required for apoptosis because plating on bovine serum albumin-blocked poly-L-lysine (allows attachment, but not integrin ligation and cell spreading) also induced apoptosis. Latrunculin B (LatB), which inhibits F-actin polymerization, induced transient loss of HBMEC spreading on vitronectin, but not their detachment, and induced apoptosis despite recovery of cell spreading. However, LatB did not cause apoptosis in 5 tumor cell lines. In HBMECs, both LatB and RGDfV induced transient Y412 and Y245 phosphorylation of endogenous c-Abl, a nonreceptor tyrosine kinase that reciprocally regulates F-actin. LatB also induced nuclear translocation of c-Abl in HBMECs. STI-571 (imatinib), a targeted therapy for BCR-ABL1(+) leukemias and inhibitor of c-Abl, platelet-derived growth factor receptor, and c-Kit, decreased endothelial apoptosis. LatB-induced HBMEC apoptosis, and its inhibition by STI-571 also occurred in a 3-dimensional collagen model, supporting physiologic relevance. Last, siRNA to c-Abl (but not nonspecific siRNA) also inhibited RGDfV- and LatB-induced apoptosis. Thus, endogenous c-Abl mediates endothelial apoptosis induced by inhibition of integrins alphavbeta3/alphavbeta5 or by LatB-induced disruption of F-actin.

PID1 increases chemotherapy-induced apoptosis in medulloblastoma and glioblastoma cells in a manner that involves NFκB

Phosphotyrosine Interaction Domain containing 1 (PID1; NYGGF4) inhibits growth of medulloblastoma, glioblastoma and atypical teratoid rhabdoid tumor cell lines. PID1 tumor mRNA levels are highly correlated with longer survival in medulloblastoma and glioma patients, suggesting their tumors may have been more sensitive to therapy. We hypothesized that PID1 sensitizes brain tumors to therapy. We found that PID1 increased the apoptosis induced by cisplatin and etoposide in medulloblastoma and glioblastoma cell lines. PID1 siRNA diminished cisplatin-induced apoptosis, suggesting that PID1 is required for cisplatin-induced apoptosis. Etoposide and cisplatin increased NFκB promoter reporter activity and etoposide induced nuclear translocation of NFκB. Etoposide also increased PID1 promoter reporter activity, PID1 mRNA, and PID1 protein, which were diminished by NFκB inhibitors JSH-23 and Bay117082. However, while cisplatin increased PID1 mRNA, it decreased PID1 protein. This decrease in PID1 protein was mitigated by the proteasome inhibitor, bortezomib, suggesting that cisplatin induced proteasome dependent degradation of PID1. These data demonstrate for the first time that etoposide- and cisplatin-induced apoptosis in medulloblastoma and glioblastoma cell lines is mediated in part by PID1, involves NFκB, and may be regulated by proteasomal degradation. This suggests that PID1 may contribute to responsiveness to chemotherapy.

Abstract 3331: PID1, a candidate tumor suppressor in pediatric and adult brain tumors, is a novel interacting partner for a cell surface receptor

BACKGROUND: PID1 is higher in fat from obese patients and is an inhibitor of insulin receptor signaling. We reported that PID1 is a novel inhibitor of medulloblastoma and glioma cell growth, and its mRNA level is highly correlated with survival across multiple independent datasets of these tumors. PID1 harbors a phosphotyrosine binding domain and is predicted to interact with proteins harboring NPXY motifs. We therefore hypothesized that PID1 will interact with phosphotyrosine-binding (PTB) domain containing protein(s) that may be important in brain tumors, and will modulate its/their signaling. Here we report PID1 interactions with one of these proteins and determine its biological consequences. METHODS: Experiments consisted of immunoprecipitations, transient transfections, and western blotting. Functionally, assays included cell culture, flow cytometry for proliferation using BrdU/7AAD and apoptosis using AnnexinV/7AAD. Plasmids were pCMV-AN-HA-PID1(2) or control or the protein of interest in pcDNA3.1. Cell lines used were glioblastoma, medulloblastoma, atypical teratoid rhabdoid (ATRT), HEK293, and cells deficient or intact in PID1. RESULTS: We demonstrate co-immunoprecipitation of PID1 with a receptor that is considered important in cancer. Immunoprecipitation was both in HEK293T cells overexpressing both proteins and in native brain tumor cell lines with/without overexpression. While tyrosine in the NPXY motifs was not required for the interaction, mutating the kinase domain of the receptor or overall inhibition of tyrosine phosphorylation impaired the interaction with PID1. Different domains in PID1 differentially co-immunoprecipitated with the receptor. Interestingly, mouse embryo fibroblasts without PID1 showed increased growth factor-dependent tyrosine phosphorylation of this protein and increased growth factor-induced proliferation. Identity of the receptor will be disclosed at the meeting. CONCLUSIONS: These data point to PID1 as a novel interacting partner and regulator of this receptor’s biological effects and signaling in brain tumors. Note: This abstract was not presented at the meeting. Citation Format: Anat Erdreich-Epstein, Xiuhai Ren. PID1, a candidate tumor suppressor in pediatric and adult brain tumors, is a novel interacting partner for a cell surface receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3331. doi:10.1158/1538-7445.AM2017-3331

Abstract 3975: PID1, a new growth-inhibitory gene, sensitizes brain tumor cell lines to chemotherapy

BACKGROUND: Phosphotyrosine Interaction Domain containing protein-1 (PID1) is a gene discovered in 2006 in the context of obesity, which we recently showed inhibits growth of brain tumor cell lines and its mRNA level is directly correlated with survival of patients with gliomas and medulloblastomas (Erdreich-Epstein et al, Clin Cancer Res, In Press). PID1 mRNA was lower in more aggressive medulloblastomas and gliomas, and patients whose tumor had higher PID1 mRNA had longer survival. Moreover, overexpression of PID1 markedly decreased 2D colony formation in cell lines of medulloblastomas, GBM and atypical teratoid rhabdoid tumors (ATRT). The growth-inhibitory effect was due to both decreased proliferation and increased cell death. METHODS/RESULTS: COSMIC showed no mutations of PID1 in brain tumors, suggesting that PID1 was not a formal tumor suppressor gene. Therefore, we hypothesized that PID1 may sensitize brain tumor cells to therapy, and thus account for the longer survival in patients with higher PID1 mRNA. Indeed, while both cisplatin (10µg/ml) or transient PID1 overexpression increased apoptosis of glioma and medulloblastoma cell lines (indicated by increased AnnexinV and mitochondrial depolarization), combining cisplatin with PID1 caused a markedly higher apoptosis. Moreover, knockdown of PID1 by siRNA inhibited the cisplatin-induced mitochondrial membrane depolarization and apoptosis (AnnexinV), suggesting that PID1 may be required for cisplatin-induced apoptosis. Taken together, this suggests a sensitizing effect of PID1 to chemotherapy in both glioma and medulloblastoma cell lines. Intriguingly, PID1 mRNA and protein both increased in response to etoposide (5µg/ml), vincristine (50ng/ml) or cisplatin (5µg/ml) in a time- and dose-dependent manner in the brain tumor cell lines. This chemotherapy-induced increase in PID1 mRNA was blocked by inhibitors of NFKB, suggesting that regulation of PID1 may be an NFKB-dependent mechanism. Ongoing work seeks to decipher the mechanism of these effects and test them in vivo in mouse models. CONCLUSIONS: Our data suggest that PID1 acts to sensitize glioma and medulloblastoma cells to chemotherapy, possibly explaining the correlation between higher PID1 mRNA and survival in patients. Citation Format: Jingying Xu, Xiuhai Ren, Anthony Tran, Gregory M. Shackleford, Anat Erdreich-Epstein. PID1, a new growth-inhibitory gene, sensitizes brain tumor cell lines to chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3975. doi:10.1158/1538-7445.AM2014-3975