Background: Current chemotherapeutics are inefficient as ovarian cancer (OVCA) patients develop resistance. Immunotherapy is safe and offers long-term prevention. However, lack of information on the interaction of immune cells and the tumor are significant barriers to the development of successful immunotherapies for OVCA. OVCA is disseminated mainly by peritoneal diffusion rather, thus, interaction between the tumor and the immune cells in the tumor vicinity are critical. Natural killer (NK) cells, members of innate immunity, are the first responders against a developing tumor. NK cells kill tumor cells by binding of its receptor, NKG2D, with ligands including MHC Class I polypeptide related sequences A and B (MICA/B), expressed on the surface of tumor. Tumor escapes recognition by NK cells by shedding MICA/B. However, the molecular mechanism(s) of tumor induced shedding of MICA/B and suppression of NK cell function is not known. Tumor associated stress factors including GRP78 and ADAM10 (a proteolytic agent), expressed on the surface of tumor cells, may be involved in shedding of MICA/B. Information on this mechanism will lead to the development of effective NK cell based immunotherapy against OVCA.

Objective: The goal of this study was to determine the molecular mechanism of suppression of NK cells by ovarian tumors and factors regulating this suppression and their possible prevention.

Materials and methods: Two studies were conducted. Exploratory: Archived ovarian malignant tumors (papillary serous, n= 7 early stage and n= 15 late stage) were examined for the expression of NK cells, GRP78, ADAM10 and MICA. Prospective: 4-years old laying hens with or without early stage OVCA were selected by ultrasound scanning, divided into 3 groups, each with 15 normal and 5 OVCA hens. One group received control diet (basal), 2nd and 3rd groups received diet supplemented with 1% or 2% Withaferin A containing root powder of the herb Ashwagandha (ASH, Withania somnifera), respectively, for 90 days. Serum samples were collected before the start and at the end of the study. Animals were sacrificed, gross morphology and tumor stages were recorded. Normal or ovaries with cancer were processed for immunohistochemistry, proteomics and gene expression studies.

Results: Compared with normal ovaries, the frequency of NK cells, intensity of expression of GRP78 and ADAM10 were significantly higher in OVCA patients at early and stages. However, NK cell frequency between the early and late stages was not significantly different. Similar patterns were also observed in control hens with OVCA without ASH treatment. In hens treated with 2% ASH, the tumor remained limited to the ovary in 4 out of 5 hens (80%) while they progressed to late stages in control hens. The expression of GRP78 and ADAM10 was significantly reduced in 2% ASH treated hens than the control and 1% treated hens, but the frequency of intra-tumoral NK cells increased. ASH treatment of hens increased the expression of miR-180a, regulator microRNA of GRP78. Decrease in GRP78 may suggest reduced stress and decreased expression of ADAM10 leading to decreased shedding of MICA/B allowing more NK cells to recognize the tumors. Thus, ASH treatment reduced the stress and expression of ADAM10 through decreased GRP78, prevents suppression of NK cells as more MICA will remain on the surface of tumor cells.

Conclusion: The results of this study suggest that enhanced expression of GRP78 in association with ADAM10 may be one of the mechanisms of suppression of NK cells. Thus, GRP78 and ADAM10 offer novel targets for immunotherapy. ASH an anti-stress natural product prevents tumor induced suppression of NK cells by reducing stress. ASH is available over the counter and may be useful as immunotherapy for preventing OVCA.

Partial support: DOD, W81XWH-14-1-0315.

Citation Format: Animesh Barua, Pincas Bitterman, Sameer Sharma, Sanjib Basu, Janice M. Bahr. Ovarian tumor-induced immunosuppression of NK cells and its prevention by dietary supplementation of herbal Withaferin A (Ashwagandha). [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 A48.