Abstract A28: Rapamycin-upregulated miR-29b promotes mTORC1-hyperactivative cell growth by downregulating retinoic acid receptor β (RARβ)
Authors: Liu Heng-Jia , Lam Hilaire C. , Baglini Christian V. , Conttrill Alex K. , Chan Stephen Y. , Henske Elizabeth P. 
Published: December 2020

Tuberous sclerosis complex (TSC) is an incurable multisystem disease associated with hamartomatous tumors of brain, heart, skin, kidney and lung. The TSC proteins (TSC1 and TSC2) inhibit the mechanistic/mammalian target of rapamycin complex 1 (mTORC1), an exquisitely sensitive molecular sensor that regulates cell growth, cell metabolism and autophagy. mTORC1 is hyperactive in many malignancies, including TSC. Treatment of TSC-associated tumors with mTOR inhibitors leads to partial responses, with prompt regrowth upon treatment cessation, highlighting the unmet clinical need for cytocidal therapies. We previously discovered that miR-29b expression is strongly upregulated following rapamycin treatment. However, the functional significance and therapeutic impact of miR-29b induction by rapamycin in the therapy of TSC is unknown. We found that miR-29b expression is induced by rapamycin in vitro in 4 different TSC2-deficient cells and in vivo TSC-2 deficient tumors. Inhibition of miR-29b significantly decreased anchorage-independent cell growth by 40% (p &lt; 0.01), and combined rapamycin treatment and miR-29b inhibition further suppressed growth by 73% (p &lt; 0.0001). In addition, miR-29b knockdown inhibited cell migration and invasion by 68% (p &lt; 0.05) and 50% (p &lt; 0.01), respectively. miR-29b inhibition combined with rapamycin further decreased cell migration and invasion by 77% (p &lt; 0.001) and 74% (p &lt; 0.001), respectively. These findings suggest that miR-29b acts as an “onco-miR” in this context. Importantly, miR-29b knockdown in TSC2-deficient cells significantly suppressed tumorigenesis in vivo by 52% (p &lt; 0.0001). Furthermore, the combination of rapamycin and miR-29b inhibition resulted in a more robust inhibition of tumor volume compared with miR control (47% vs. 24% inhibition) (p &lt; 0.01). To gain insight into the molecular mechanisms by which miR-29b promotes tumorigenesis, we performed RNA-sequencing on miR-29b knockdown and control cells treated with rapamycin. 236 genes were upregulated in miR-29b knockdown cells by at least 2.0-fold and p &lt; 0.05. Retinoid receptor beta (RARβ), one of the candidates, was increased in miR-29b knockdown cells at the mRNA level by 2.25-fold (p &lt; 0.01) and protein expression by 2-fold (p &lt; 0.05). RARβ is a tumor suppressor in many types of tumors. Interestingly, RARβ expression is downregulated by rapamycin (67% (p &lt; 0.001)) and upregulated 4-fold following miR-29b inhibition (p &lt; 0.05). Lastly, we found the 3'UTR activity of RARβ was upregulated in miR-29b knockdown cells by 4-fold (p &lt; 0.001) using a luciferase reporter assay, supporting a model in which RARβ is a direct target of miR-29b. These studies demonstrate that miR-29b induction by rapamycin promotes TSC2-deficient tumorigenesis. miR-29b is a potential therapeutic target for TSC-associated tumors and possibly other mTORC-1 hyperactive tumors.

Citation Format: Heng-Jia Liu, Hilaire C. Lam, Christian V. Baglini, Alex K. Conttrill, Stephen Y. Chan, Elizabeth P. Henske. Rapamycin-upregulated miR-29b promotes mTORC1-hyperactivative cell growth by downregulating retinoic acid receptor β (RARβ) [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A28.