Abstract A35: Regulation of the tumor microenvironment by oncogenic p62 in mTORC1-hyperactive diseases: Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM)
Authors: Lam Hilaire C. , Parkhitko Andrey , Lope Alicia Llorente , Alesi Nicola , Khabibullin Damir , Filippakis Harilaos , Pereira Ana , Ogorek Barbara , Zhang Erik , Yu Jane , Priolo Carmen , Henske Elizabeth P. 
Published: October 2016

Abstract
Tuberous sclerosis complex (TSC) is a multisystem disease associated with tumors of the brain, skin and kidney as well as progressive cystic lung destruction characteristic of lymphangioleiomyomatosis (LAM). TSC and LAM are caused by loss-of-function mutations in TSC1 or TSC2, resulting in hyperactive mechanistic Target of Rapamycin complex 1 (mTORC1) signaling. We previously discovered that mTORC1 hyperactivation in TSC inhibits autophagy and promotes the accumulation of oncogenic p62/sequestosome 1 (PNAS, 2011). p62 exerts a pleiotropic role in tumor progression, including the regulation of NRF2 to combat oxidative stress, NFκB to promote cell survival and the degradation of ubiquitinated proteins to efficiently recycle organelles and proteins by autophagy. Knockdown of p62 in Tsc2-null cells inhibited tumorigenesis in xenografts in vivo, but did not impact proliferation in vitro, leading us to hypothesize that p62 exerts non-cell autonomous functions influencing the tumor microenvironment to promote Tsc2-/- cell tumorigenic potential.
In unpublished work, we explored the molecular mechanisms by which p62 promotes tumorigenesis in TSC/LAM. First, we analyzed renal tumor burden in Tsc2+/-; p62-/- mice. Compared to littermate control Tsc2+/-; p62+/+ mice, Tsc2+/-; p62-/- mice have a 2-fold (p&lt;0.05) and 10-fold (p&lt;0.05) reduction in macroscopic cystic index and in microscopic tumor burden, respectively. Next, p62 was downregulated in Tsc2-null cells. Downregulation of p62 reduced colony formation in soft agar by 3-fold in Tsc2-null MEFs relative to control shRNA (p&lt;0.001). Knockdown of p62 decreased Tsc2-null MEF invasion through matrigel chambers 2-fold (p&lt;0.001), while migration towards a chemoattractant (10% serum) was p62 independent. Cytokine profiing with a Luminex 32-plex assay revealed that interleukin 6 (IL6) is significantly upregulated in Tsc2-null cells compared to wildtype controls. Surprisingly, both inhibition of mTORC1 and p62 knockdown increased IL6 levels detected in the media (by 4-fold and 2-fold respectively, p&lt;0.05). mTORC1 inhibition by rapamycin and p62 knockdown both increased IL6 expression 2-fold at the mRNA level (p&lt;0.05). In parallel with these in vitro findings, we performed multiplex cytokine profiling on human LAM patient serum and discovered that IL6 is significantly upregulated compared with healthy controls (5-fold, p&lt;0.05). Interestingly treatment of p62 knockdown cells with either rapamycin (20nM, 24h) or Torin1 (250nM, 24h) further increased IL6 levels (2-fold and 4-fold respectively, p&lt;0.05). IL-6 both activates transcriptional activity of STAT3 and is positively regulated by STAT3. In Tsc2-null cells, we found a 3-fold (p&lt;0.01) increase in phosphorylation of STAT3 at Y705 relative to wildtype cells. Downregulation of p62 resulted in a 5-fold (p&lt;0.001) increase in phosphorylation at Y705 compared to wildtype cells, suggesting that p62 promotes tumorigenesis in TSC-deficient cells via regulation of STAT3 and IL6.
Our data point toward a model in which p62 and mTORC1 cooperatively regulate cellular IL6 in Tsc2-null cells to promote tumorigenesis in TSC. IL6 is correlated with poor prognosis, advanced disease and metastatic potential in numerous cancers. Rapamycin dramatically decreases p62 levels in TSC-deficient cells. Therefore, the induction of IL6 by rapamycin through decreased p62 expression may represent one of the first non-cell autonomous mechanisms by which mTORC1 inhibition impacts tumorigenesis. Since mTORC1 is hyperactivated in the majority of malignant tumors and more than 100 cancer clinical trials are ongoing with mTORC1 inhibitors, our findings may yield insights with broad therapeutic potential.
Citation Format: Hilaire C. Lam, Andrey Parkhitko, Alicia Llorente Lope, Nicola Alesi, Damir Khabibullin, Harilaos Filippakis, Ana Pereira, Barbara Ogorek, Erik Zhang, Jane Yu, Carmen Priolo, Elizabeth P. Henske. Regulation of the tumor microenvironment by oncogenic p62 in mTORC1-hyperactive diseases: Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM). [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A35.