Abstract PR18: Survival of ARHI-induced dormant autophagic cell death in vivo requires permissive levels of VEGF, IL-8, and IGF-1 in the tumor microenvironment.
Authors: Lu Zhen , Orozco Aaron F. , Yang Hailing , Sutton Margie N. , Wang Yan , Mao Weiqun , Levine Douglas A. , Bast Robert C. , Bast Robert C. 
Published: May 2016

Abstract
Among gynecological cancers, ovarian cancer has the worst prognosis. One of the most important factors contributing to poor outcomes is the ability of drug resistant ovarian cancer cells to remain dormant for years after treatment, only to grow progressively and kill their host. Judging from sites of recurrence, most dormant ovarian cancer cells are found on the surface of the peritoneal cavity, often in small, poorly vascularized collagenous scars observed at second look operations. Our group has found that autophagy and tumor dormancy can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), which is downregulated in 60% of ovarian cancers and in cancers from several other sites. Re-expression of ARHI induces autophagy by inhibiting PI3K and mTOR signaling, displacing Bcl-2 from Beclin1to form the autophagy initiating complex, inducing ATG4, decorating the autophagosome membrane and co-localizing with LC3II. Re-expression of ARHI in ovarian cancer cells in culture produces cell death within 72 hours, whereas re-expression of ARHI in xenografts produces cell growth arrest and tumor dormancy. When ARHI levels are reduced once again in vivo, xenografts grow promptly to kill the host. Outgrowth of dormant xenografts is markedly delayed, but not prevented by treatment with chloroquine, a functional inhibitor of autophagy. The clinical relevance of this dormancy model is supported by previous studies that found ARHI expression in ovarian cancer cells was significantly increased in 90% of cases where small, dormant nodules of drug resistant ovarian cancer removed from the peritoneal surface at second-look operations, but in only 40% of the primary specimens from the same patients from initial cytoreductive surgery. In this study, we have measured Ki67 and microvascular density (MVD) in the same set of primary human ovarian cancers and in matched positive second look specimens. Second look specimens exhibited significantly lower Ki-67 (p&lt;0.0001) and MVD (p&lt;0.0001) than did primary cancers. We have hypothesized that that decreased access to nutrients with decreased MVD could be responsible the elevation of ARHI levels and the induction of autophagy. While autophagy could sustain nutrient deprived cancer cells up to a point, excessive autophagy can also induce type II autophagic cancer cell death. Our previous studies indicated that growth factors (VEGF, IGF1) and cytokines (IL-8) present in the tumor microenvironment could rescue ovarian cancer cells in culture from autophagic death. In this study we have found that treatment with anti-VEGF, anti-IL8 and anti-IGFR1 antibodies, individually and in combination, slowed the outgrowth of dormant autophagic ovarian cancer cells. Treatment of dormant xenografts with the same monoclonal antibodies against VEGF, IL-8 and IGFR1 with or without CQ not only delayed outgrowth when DOX is withdrawn, but cured a fraction of mice. Re-expression of ARHI reduced expression of IGF-1 receptor, but not VEGF and IL8 receptors. Treatment ovarian cancer cells with VEGF, IL-8 and IGF-1 reduced ARHI's ability inducing autophagy and inhibiting AKT and mTOR activity. Our studies provide a novel model for tumor dormancy that incorporates autophagy as a mechanism for tumor cell survival in the presence of permissive levels of growth factors and cytokines. Blocking these survival factors with antibodies could provide a novel approach to eliminate dormant cells, providing more effective treatment for women with ovarian cancer.
This abstract is also presented as Poster A36.
Citation Format: Zhen Lu, Aaron F. Orozco, Hailing Yang, Margie N. Sutton, Yan Wang, Weiqun Mao, Douglas A. Levine, Robert C. Bast, Jr., Robert C. Bast, Jr.. Survival of ARHI-induced dormant autophagic cell death in vivo requires permissive levels of VEGF, IL-8, and IGF-1 in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr PR18.