Abstract P5-10-04: Exploring novel connections between dishevelled (DVL) proteins and HER2-positive breast cancer-road to translation
Authors: Rasha Fahmida , Yang Mingxiao V , Castro-Piedras Isabel , Furr Kathryn L. , Snitman Annie , Khan Sonia Y. , Brandi Luis , Khan Hafiz , Jahan Nusrat , Melkus Michael W. , Pruitt Kevin , Rahman Rakhshanda Layeequr 
Published: March 2022

Aberrant Wnt signaling contributes to every stage of tumorigenesis from immunosuppression in the tumor microenvironment (TME) and reduced tumor infiltrating lymphocytes (TIL) activity in the tumor niche to modulating several cancer hallmarks such as unrestrained growth, increased neoplastic cell survival, invasion, and metastasis. The overactive Wnt pathway is critical for tumor progression and hence a therapeutic target in multiple clinical trials. Dishevelled (DVL)s are the major scaffold proteins which relay signals in both canonical and non-canonical Wnt pathways and help maintain constitutive oncogenic signaling. Though DVL proteins are significant drivers of oncogenic Wnt signaling, little is known about their role in modulating tumor immunity. Previously we demonstrated that nuclear localization of DVL proteins is associated with transcription of several cancer-associated genes as well promotion of in vivo tumor growth. Moreover, our preliminary studies revealed significant positive association between high DVL expression and poor patient survival in different aggressive subtypes of breast cancer including HER2-positive breast cancer. Therefore, we hypothesize that (i) DVL depletion will reduce expression of classic Wnt markers involved in cancer cell proliferation and invasion in HER2-overexpressed in vitro models of breast cancer and (ii) clinically, high DVL protein expression will modulate markers of tumor immunity such as TIL score, levels of cytotoxic (CD8α) T-cells, neutrophil/platelet to lymphocyte ratio (NLR, PLR), eventually resulting in poor survival outcomes in HER2-positive breast cancer.To test our hypothesis, we performed DVL2 loss of function studies in two different HER2-overexpressed breast cancer cell lines such as SKBR3 (ER/PR-, HER2+) and BT474 (ER/PR+, HER2+) via lentiviral shRNA transduction and siRNA transfection. We analyzed RNA (RTqPCR) and protein (western blot) expression of classic Wnt markers and performed cell proliferation and cell cycle analyses by live cell imaging and flow cytometry, respectively. Additionally, we performed a pilot study including 10 HER2-positive breast cancer patients to dissect the role of DVL2 in tumor immunity. Retrospective chart review on patient records and banked tissue histology were performed to investigate the markers of tumor immunity and DVL proteins. Data were analyzed in SPSS (version 25) and GraphPad Prism (version 7) at a significance p&lt;0.05.Initially, we identify higher DVL2 protein levels in tissues from HER2-positive breast cancer compared to non-cancer and other subtypes of breast cancer. Next, DVL2 depletion (NTC vs. sh- or siDVL2) results in reduced mRNA expression of Wnt target genes such as CYCLIN D1, OCT4, MMP7, VEGFA classically involved in cell proliferation, stem cell renewal, migration, and invasion respectively in both SKBR3 and BT474 cells. Similarly, live cell proliferation and cell cycle analyses reveal that DVL2 knockdown resulted in reduced proliferation, higher growth arrest (G1), limited mitosis (G2/M) compared to non-targeted control. Analyses on patient tissues further demonstrate that higher DVL2 expression pose a weak negative correlation with % TIL score (r=-0.17, NS) and % CD8α levels (r=-0.32, NS) while have a positive correlation with NLR (r=0.65, p=0.05), where high NLR denotes worse cancer prognosis. Though not statistically significant, these results from our pilot study reveal crucial roles of DVL2 proteins in regulating immunity and survival of HER2-positive breast cancer patients. Our study is noteworthy since it demonstrates possible immune regulatory role of DVL proteins in HER2-positive breast cancer which in turn, will aid in designing future clinical trials and in-depth mechanistic studies benefitting patients of different subtypes of breast cancer.

Citation Format: Fahmida Rasha, Mingxiao V Yang, Isabel Castro-Piedras, Kathryn L. Furr, Annie Snitman, Sonia Y. Khan, Luis Brandi, Hafiz Khan, Nusrat Jahan, Michael W. Melkus, Kevin Pruitt, Rakhshanda Layeequr Rahman. Exploring novel connections between dishevelled (DVL) proteins and HER2-positive breast cancer-road to translation [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-04.