Abstract 91: Classification of cancer cells based on unexpected variation in levels and dynamics of pRb, pERK, and pAKT in response to cell synchronization from nutrient deprivation

Abstract

Abstract The tumor suppressor Rb and the RAS-RAF-MEK-ERK and PI3K-AKT-mTOR signaling cascades are often dysregulated in cancer. As insufficient vasculature in the tumor microenvironment (TME) leads to nutrient deficiency, cellular responses to nutrient deprivation is relevant to consider in the treatment of solid tumors. Here, we demonstrate that levels of pRb, pERK, and pAKT after cell synchronization via nutrient deprivation and after release via serum stimulation vary unexpectedly across a panel of nine cancer and two normal cell lines. From these results, we describe a novel classification of tumor cells defined by their behavior in nutrient-limiting conditions. Four colorectal, four lung, and one breast cancer cell line, and two normal lung fibroblasts were included in the panel. A range of KRAS, NRAS, BRAF, and PIK3CA mutations as well as wild-type cells were represented. In 6-well plates, 1E5-5E5 cells were plated per well in media containing 10% serum, incubated for 16-24 hours, then were nutrient-deprived in media containing 0% or 1% serum for 2, 4, 8, 12, 24, 48, 72, or 96 hours. Synchronization was released by serum stimulation with 20% serum-containing media for 24-48 hours. Cells were imaged, counted, and harvested, then protein levels of Rb, pRb, ERK, pERK, AKT, and pAKT were measured in cell lysates using a western blot. Nutrient deprivation slowed the proliferation of each cell line. Surprisingly, phosphorylation of Rb at Ser795 and Ser807/811 after nutrient deprivation varied across cell lines. pRb remained at baseline in MCF7 cells, decreased in RKO and H1299 cells, and increased in HCT116 cells. Despite this, levels of the pRb upstream regulator CDK6 decreased consistently across these cell lines, suggesting possible alternate regulation of pRb in MCF7 and HCT116 cells. Unanticipated variation in pERK levels after nutrient deprivation was also observed, including a dramatic increase in pERK upon starvation in HCT116 cells but not in others. Similar notable findings were made as far as pAKT. pAKT levels changed dynamically across time points in some cell lines, but consistently increased in some (MCF7) and decreased in others (RKO). Cell synchronization release via serum stimulation caused varying levels of pRb and pERK restoration. Our novel findings reveal a broad new classification of tumor cells defined by their behavior nutrient-limiting conditions. Such classification may guide the use of nutrient deprivation as a predictive biomarker for CDK, MEK, or mTOR inhibitors in solid tumors with nutrient-poor TMEs, as we are currently exploring. We are also evaluating the impact of nutrient deprivation on immune cell killing and cytokine secretion of specific classes of cancer cells that we define here, and are working to establish real-time cell cycle markers to visualize differential effects of serum deprivation and stimulation. Citation Format: Lindsey Carlsen, Wafik S. El-Deiry. Classification of cancer cells based on unexpected variation in levels and dynamics of pRb, pERK, and pAKT in response to cell synchronization from nutrient deprivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 91.