Abstract The defining feature of premalignant mammary lesions is proliferation of cells within the intraductal niche. Survival in this stressful duct microenvironment requires sophisticated mechanisms to avoid apoptosis caused by hypoxia, nutrient deprivation, metabolic acidosis, and intracellular calcium overload. Two major survival mechanisms of breast ductal carcinoma cells (DCIS) are 1) the upregulation of autophagy and 2) the enhanced export of calcium via plasma membrane calcium-ATPase isoform 2 (PMCA2) efflux pumps. We hypothesize that calcium efflux, through PMCA2, and activation of autophagic catabolism are critical interdependent mechanisms for the survival of pre-invasive human breast neoplastic cells. To assess the proteomic response to autophagy inhibition and/or elevated intracellular calcium, 41 signal transduction proteins/post-translationally modified proteins related to proliferation, autophagy, and calcium pathways were quantified by reverse phase protein microarrays (RPPA) in time course studies of T-47D metastatic ductal breast cancer cell line, and MCF-10A non-tumorigenic breast cell line, both of which express low basal levels of PMCA2, and in human primary DCIS organoid cultures. Cell lines were treated with chloroquine (50μM), or 1,25 dihydroxyvitamin D3 (Calcitriol) (50nM or 100nM), or chloroquine + Calcitriol, and harvested at periodic intervals over 197 hours. Stoichiometric time course studies (24 hours) were evaluated for Calcitriol as a single agent in both T-47D and MCF-10A cell lines. Cell morphology, adhesion, proliferation and autophagic flux in the DCIS organoids were assessed by Immunohistochemistry and RPPA. Calcitriol alone did not increase proliferation based on PCNA expression within DCIS ducts. Chloroquine markedly increased Cleaved PARP (Chi Sq p = 0.004) and CAMKinase II (p = 0.0068). Chloroquine (50μM) plus Calcitriol (100nM) increased E-Cadherin (p = 0.0379) and phosphoPAK1/2 (p = 0.0163), while PMCA2 levels were not significantly different between treatment groups. Autophagy inhibition and calcium efflux may be exploited therapeutically by co-administration of chloroquine and Calcitrol, to inhibit lysosomal catabolism and increase intracellular calcium levels, respectively, switching them from autophagic survival into apoptotic death. Citation Format: Virginia A. Espina, Solomon Yeon, Joshua N. VanHouten, John Wysolmerski, Lance A. Liotta. Chloroquine and vitamin D3 modulate proliferation in early stage breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2859.