The activating immune ligands, MICA/B, act as a "kill me" signal through the NKG2D receptor expressed on natural killer (NK) cells. Recently, the oncogenic miR-20a was found to mediate immune escape through repressing MICA/B levels in breast cancer (BC) cells. However, targeting miR-20a-MICA/B using natural compounds has rarely been investigated. Our group has successfully isolated 3'-O-acetylvitexin that showed cytotoxic effects against colon cancer cells but has never been evaluated in BC. Our aim is to investigate the effects of 3'-O-acetylvitexin on BC cell lines and to further elucidate its molecular mechanism of action. The results showed that 3'-Oacetylvitex in depicted a more pronounced dose-dependent repression of TNBC cellular viability, colonogenicity and migration capacity than Vitexin. 3'-O-acetylvitexin treatment resulted in a marked dose-dependent repression of miR-20a with a concomitant dose-dependent increase in MICA/B expression. In conclusion, 3'-O-acetylvitexin might act as a promising therapeutic agent for TNBC patients.

Background: A Palestinian woman with breast cancer only has 50% chance of being alive at five years. Lack of access to medical care is further complicated by political strife, checkpoints, and the need to file a permit for traveling even a short distance. Preliminary studies of Palestinian women under 40 with breast cancer show a low frequency of BRCA1/BRCA2 mutations. These studies suggest that there may be unique genetic, epigenetic, and environmental drivers of aggressive cancers in Palestinian women. Here we have developed the Palestinian infrastructure to start to investigate these drivers. We report our efforts to build infrastructure and our initial findings. Methods: To build capacity, we first trained 2 Palestinians in genetic counseling using the City of Hope Distance Learning Program. We engaged a group of Palestinian breast surgeons from the cities of Ramallah, Jerusalem, Nabulus, and Hebron. Together we built the infrastructure for blood and tissue collection/preservation. Our protocol was first approved by the Palestinian Ministry of Health and Palestinian Helsinki Committee. To date we have enrolled 65 Palestinian women from throughout Palestine and collected matched breast cancer tissue and matched white blood cells (WBC) DNA. On WBC DNA we performed whole-exome sequencing (WES) to search for germline mutations. Exomes were enriched using Agilent SureSelectHumanAllExonV6+COSMIC V2 kit, which covers 66 MB of the genome. Any deleterious mutation will be formally verified by MyriadTM United States CAP-CLIA-approved testing. Results: The average age of the 65 women was 44 (range 18-78). Women were tested for mutations in 20 genes known to be associated with DNA repair and breast cancer. Of these 65 women, none had a family history of breast cancer; to date, no known pathogenic variants have been identified in these women. Parallel studies in a second group of Palestinian women with a history of familial breast cancer had pathogenic mutations in 11 different genes: BRCA1, BRCA2, TP53, ATM, CHEK2, BARD1, BRIP1, PALB2, MRE11A, PTEN and XRCC2. The single most frequent mutation was TP53 p.R181C, which was significantly enriched in the discovery series compared to controls (p50.01) and was responsible for 15% of breast cancers among young onset or familial patients. Conclusions: The genetic and possibly epigenetic origins of breast cancer in Palestinian women may be distinct from non-Hispanic White Women. We have formed a Palestinian Coalition to ethically obtain matched WBC and tumor tissue from Palestinian women with breast cancer. Our studies show that in accordance with Helsinki Principles, we have developed the infrastructure to return testing results to the referring physicians. Note: This abstract was not presented at the conference. Citation Format: Sundus Shalabi, Jerneja Tomsic, NourAl-Huda Hamad, Susan Neuhausen, Osama Atallah, Saeed Mustafa, Akram AbuHeibeh, Omar Abdul-Shafi, Nufuz Maslamani, Bashar Karmi, Ola Abu-Kishik, Jehad Shawar, Haifa Aqeilan, Aya Awad, Rami Aqeilan, Ahmad Dalbah, Hanood Abras, Abdul-Rahman Salamheh, Firas Jallad, Hisham Darwish, Victoria Seewaldt. Whole-exome sequencing of DNA from Palestinian women undergoing surgical evaluation for breast cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C057.

Background: The activating immune ligand, MICA, acts as a "kill me" signal through the NKG2D receptor expressed on natural killer (NK) cells that are key players in the fight against breast cancer (BC). Shedding of MICA during BC progression acts as a formidable barrier against NK cells' immune-surveillance. Recently, miR-20a was found to mediate immune escape through repressing MICA levels on BC cells. However, targeting miR-20a/MICA using natural compounds has seldomly been investigated. Vitexin, a flavone Cglycoside, showed potent anticancer properties. It was reported that acetylation of glycosides increases their cytotoxic activity, with an unknown impact on immunogenicity. Our group has successfully isolated 3'-O-acetylvitexin from Ocimum basilicum which showed potent cytotoxic effects against colon cancer cells but has never been investigated in BC. Our aim is to unravel the role of the immunogenic miR-20a/MICA axis in BC patients and its regulation by vitexin and 3'-O-acetylvitexin. Methods: Breast tissues were collected from 26 BC patients. ER, PR and HER2 expression was quantified using immunohistochemistry. MDA-MB-231 TNBC cells and MCF-7 HRþ BC cells were treated with serial dilutions of vitexin and 3'-O-acetylvitexin. Their cytotoxic activities were assessed using MTT, colony forming and migration assays. Total RNA was extracted, reverse transcribed, then MICA and miR-20a were quantified using qRT-PCR. Results: miR-20a is upregulated in BC patients, while MICA was downregulated in MDA-MB-231 compared to MCF7 cells. Vitexin decreased MDA-MB-231 cellular viability and migration capacity. 3'-O-acetylvitexin resulted in a more pronounced dosedependent repression of TNBC cellular viability, colonogenicity and migration capacity. Treatment with vitexin didn't show any alteration in miR-20a but showed only 2 folds increase in MICA. However, 3'-O-acetylvitexin markedly decreased miR-20a with a concomitant increase in MICA by 12 folds. Conclusions: 3'-O-acetylvitexin displays more pronounced anticancer properties against TNBC through halting their progression and immune suppressive nature by modulating miR-20a/MICA axis. This highlights miR-20a/MICA axis as a potential therapeutic target in BC.