Ng, C. Y. | Search bioRxiv

bioRxiv2020

mTOR S-nitrosylation inhibits autophagy and lysosomal proteolysis

Mammalian Target of Rapamycin (mTOR) is a master regulator of autophagy and lysosomes, and its downstream kinase-dependent pathways have been extensively characterized. Here, we report an unexpected kinase-independent regulation of autophagy and lysosomes by S-nitrosylation at Cys423 position of mTOR that resulted in suppression of VPS34 and PIKfyve-dependent phosphoinositide synthesis. Physiologically, S-nitrosylation of mTOR reduced basal lysosomal proteolysis via nitric oxide synthase (NOS)-mediated synthesis of NO from lysosomal arginine precursor, a marker of cellular nutrition status. Significantly, we found increased lysosomal NOS-mTOR complexes in APP-PS1 Alzheimers disease (AD) murine model, and increased mTOR S-nitrosylation in AD patient-derived fibroblasts. Lastly, we demonstrated that pharmacological inhibition of NOS or overexpression of mTORCys423Ala mutant reversed lysosomal and autophagic dysfunction in AD patient-derived fibroblasts, suggesting novel therapeutic strategies for autophagosome-lysosomal activation.

cell biology↗

bioRxiv2020

Stroma transcriptomic and proteomic profile of prostate cancer metastasis xenograft models reveals conservation of bonemicroenvironment signatures

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men with therapy resistance acquisition to androgen deprivation treatment and metastasis progression. Understanding the mechanisms of tumor progression to metastatic stage is necessary for the design of therapeutic and prognostic schemes. The main objective of the current study is to determine, using transcriptomic and proteomic analyses on patient derived-xenograft models, whether differentially aggressive PCa tumors predispose their microenvironment (stroma) to a metastatic gene expression pattern, and how this information could be applied in prognostics. Transcriptomic profiling (RNA Sequencing) was performed on PCa PDX models representing different disease stages; BM18 (androgen dependent bone metastasis) and LAPC9 (androgen independent bone metastasis). Using organism-specific reference databases, the human-specific transcriptome, representing the tumor, was identified and separated from the mouse-specific transcriptome (representing the contributing stroma counterpart) from the same PDX tumor samples. To identify proteome changes in the tumor (human) versus the stroma (mouse), we performed human and mouse cell separation using the MACS mouse depletion sorting kit, and subjected protein lysates to quantitative TMT labeling and mass spectrometry. We show that tenascin C is one of the most abundant stromal genes in bone metastasis PCa PDXs, is modulated by androgen levels in vivo and is highly expressed in castration resistant LAPC9 PDX compared to castration sensitive BM18 PDX. Tissue microarray of primary PCa samples (N=210) was used to evaluate the potential of TNC to act as a metastasis prognosis marker. Low number of TNC-positive cells were associated with statistically significant clinical progression to local recurrence or metastasis, compared to high TNC-positive group. Our data showed that metastatic PCa PDXs that differ in androgen sensitivity trigger a differential stroma response suggesting that stroma was influenced by tumor cues. Selected stromal markers of osteoblastic PCa induced bone metastases, were induced in the microenvironment of the host organism in metastatic xenografts, although implanted in a non-bone site, indicating a conserved mechanism of tumor cells to induce a stromal pre-metastatic signature with high potential prognostic or diagnostic value.

cancer biology↗