The Mechanism by which TPC2 Mediates Autophagy Progression and Exosome Secretion in Cancer Cells


Student thesis: Doctoral Thesis

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Award date29 Jun 2020


Autophagy is an evolutionarily conserved lysosomal degradation process, and is involved in various cellular processes. Here I studied the role of two pore channel 2 (TPC2), a lysosomal non-selective Na+/Ca2+ channel, in autophagy progression. I found that TPC overexpression in 4T1 mouse breast cancer cell line or in HeLa human cervical cancer cell line inhibited the fusion between autophagosome and lysosome, resulting in the accumulation of autophagosomes accompanied with increased lysosomal pH. On the other hand, TPC2 knockdown in 4T1 cells resulted in the increases of autolysosomes. Interestingly, I also found that exosome secretion was markedly induced in TPC2- knockdown cells but was inhibited in TPC2-overexpressing cells. Although exosome uptake was marginally increased in TPC2-knockdown cells as compared to that in control 4T1 cells, its uptake in TPC2-overexpressing HeLa cells was significantly decreased as compared to that in control HeLa cells. In addition, I found that the migration of TPC2-knockdown 4T1 cells was significantly slower as compared to that of control 4T1 cells. TPC2-knockdown cells also proliferated slower than control or TPC2-overexpressing 4T1 cells. Therefore, TPC2-knockdown cells might reduce the cell migration through the slower growth rate.

In order to explore the molecular mechanisms underlying TPC2-mediated autophagy regulation, I applied the BioID method to identify TPC2 interacting proteins. By analyzing the mass spectrometry result, I found that LAMTOR1 (late endosomal/lysosomal adaptor MAPK and mTOR activator1), which is a novel adaptor of the lysosomal membrane, is a potential TPC2 binding protein. By co-immunoprecipitation, I found that LAMTOR1 interacted with TPC2. I also found that LAMTOR1-knockdown not only induced the early stage of the autophagy, but also inhibited the autophagosome fusion with the lysosome. I speculated that LAMTOR1 knockdown inhibits the mTOR activity to induce the early stage of autophagy through TPC2 and alkalizes the pH of the lysosome to inhibit the fusion process. Thus, LAMTOR1 appears to act upstream of TPC2 to regulate autophagy. Taken together, these results support a role of TPC2 in autophagy progression and vesicle trafficking in cancer cells.