Investigation Of The Effects Of Nutrient Restriction On Endolysosomal Signaling In Colorectal Cancer (Crc) Cells.

Abstract Cancer cells grown under the stress of nutrient restriction can rewire endo-lysosomal activation and initiate autophagy as a survival mechanism. Colorectal cancer (CRC) cells are known to be particularly susceptible to the restriction of specific nutrients such as glucose and glutamine; nonetheless this susceptibility can vary considerably between cell lines. Caco-2, T84 and LoVo CRC cell lines were cultured for 2h - 48h in a pre-optimized starvation medium containing low serum, glucose and L-glutamine and pathways associated with autophagy, as well as lysosomal trafficking, biogenesis and degradation were examined in these cells. With nutrient restriction, all three cell lines showed induction of autophagy via an increase in LC3-II and p-AMPK. However, autophagic flux was inhibited in starved T84 and LoVo cells reflected by an increase in the cargo protein p62; starved Caco-2 cells showed normal autophagic flux. We therefore hypothesized a recalibration of nutrient sensing signals via the endo-lysosomal pathway in T84 and LoVo cells under nutrient restricted conditions. Nutrient restricted LoVo cells showed a surprising increase in mTORC1 activity and a decrease in the mRNA expression of MCOLN1, a cation-permeable channel on lysosomes and a transcriptional target of the master transcription regulator of lysosomal biogenesis Transcription factor EB (TFEB). Nutrient restricted T84 and Caco-2 cells showed a decrease in mTORC1 activity and a modest increase in MCOLN1 expression. A remarkable increase in the protein expression of early (Rab5) and late (Rab7) endosomal markers was observed in LoVo and T84 but not in Caco-2 cells. Additionally, treatment of both unstarved and starved T84 and LoVo cells with the v-ATPase inhibitor Bafilomycin A1 (Baf) led to an accumulation of Rab5 and Rab7; this accumulation was reversed in unstarved but not in starved cells upon withdrawal of Baf. The mRNA expression of Rab5 and Rab7 either decreased or remained unchanged in the starved T84 cells, implicating either post-transcriptional regulation via miRNAs or enhanced protein synthesis. Small RNA sequencing and subsequent confirmation with RT-qPCR of nutrient restricted cells indicated the differential expression of a network of miRNAs targeting protein involved in endo-lysosomal signalling. Additionally, the activation of the mTORC1 pathway is strongly suggestive of enhanced protein synthesis in response to the stress of starvation. Overall, our data suggests that nutrient restriction can lead to a highly rapid deregulation in endo-lysosomal signalling via post-transcriptional mechanisms. Future studies will indicate whether such deregulation provides a survival advantage to cancer cells and whether perturbation of nutrient sensing pathways can be used as a therapeutic option. Citation Format: Aliye E. Gulec, Hepsen H. Husnugil, Goksu Oral, Ilir Sheraj, Sreeparna Banerjee. Investigation of the effects of nutrient restriction on endolysosomal signaling in colorectal cancer (CRC) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1983.