Knockdown Of Rictor Research Articles

Abstract PO-113: Molecular interplay between dormant bone marrow-resident tumor cells (BMRCs) and CTCs in breast cancer

Abstract Despite widespread knowledge that bone marrow-resident breast cancer cells (BMRCs) affect tumor progression, signaling mechanisms of BMRCs implicated in maintaining long-term dormancy have not been characterized. Further, there is no model of clinical dormancy that can comprehensively emulate the entire spectrum of heterogeneous clones of Circulating Tumor Cells (CTCs) present in cancer patients. To overcome these hurdles, we developed a new experimental model of clinical dormancy employing patient-isolated de novo CTCs and their injection in xenografts with subsequent tumor monitoring and CTC characterization (ex vivo CTCs). We hypothesized that significant distinctions exist between signaling pathways of bone marrow-homing vs metastasis-competent CTCs upon transplantation in xenografts. Comparative transcriptomic analyses of ex vivo vs de novo CTCs identified increased mTOR signaling – a critical pathway frequently dysregulated in breast cancer and implicated in cell survival and dormancy – with contrasting actions by its two complementary arms (mTORC2/mTORC1). Heightened mTORC2 downstream targets augmented quiescent CTCs (Ki67-/RBL2+ cells) in paired breast cancer tissues, along with high mTORC2 activity in solitary BMRCs and tissue-resident CTCs. Further, shRNA mediated knockdown of RICTOR, an essential component of mTORC2, augmented Ki67/PCNA biomarker expression and proliferation. Collectively, these findings suggest that the balance between mTORC1 vs mTORC2 signaling regulates CTC-associated mitotic and/or dormancy characteristics. Further elucidation of mTOR-mediated CTC dormancy will provide novel strategies for therapeutic interventions in patients having early-event metastatic seeding but yet to develop clinically-detectable overt metastasis. Citation Format: Debasish Boral, Haowen N. Liu, S. Ray Kenney, Dario Marchetti. Molecular interplay between dormant bone marrow-resident tumor cells (BMRCs) and CTCs in breast cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-113.

Metabolism of short-chain fatty acid propionate induces surface expression of NKG2D ligands on cancer cells.

SCFAs are primarily produced in the colon by bacterial fermentation of nondigestible carbohydrates. Besides providing energy, SCFAs can suppress development of colon cancer. The mechanism, however, remains elusive. Here, we demonstrate that the SCFA propionate upregulates surface expression of the immune stimulatory NKG2D ligands, MICA/B by imposing metabolic changes in dividing cells. Propionate-mediated MICA/B expression did not rely on GPR41/GPR43 receptors but depended on functional mitochondria. By siRNA-directed knockdown, we could further link phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in gluconeogenesis to propionate regulation of MICA/B expression. Moreover, knockdown of Rictor and specific mTOR inhibitors implicated mTORC2 activity with metabolic changes that control MICA/B expression. SCFAs are precursors to short-chain acyl-CoAs that are used for histone acylation thereby linking the metabolic state to chromatin structure and gene expression. Propionate increased the overall acetylation and propionylation and inhibition of lysine acetyltransferases (KATs) that are responsible for adding acyl-CoAs to histones reduced propionate-mediated MICA/B expression, suggesting that propionate-induced acylation increases MICA/B expression. Notably, propionate upregulated MICA/B surface expression on colon cancer cells in an acylation-dependent manner; however, the impact of mitochondrial metabolism on MICA/B expression was different in colon cancer cells compared with Jurkat cells, suggesting that continuous exposure to propionate in the colon may provide an enhanced capacity to metabolize propionate. Together, our findings support that propionate causes metabolic changes resulting in NKG2D ligand surface expression, which holds potential as an immune activating anticancer therapy.

Abstract 1017: RICTOR/mTOR signaling regulates novel immune checkpoints in non-small cell lung cancer (NSCLC)

Abstract Background: RICTOR is a critical component of the mTOR2 complex. Through activation of AKT, SGK1, PKC and other downstream pathways, the RICTOR/mTOR signaling is essential in controlling cell survival, proliferation, metabolism, migration, protein degradation and cytoskeleton functions. Moreover, our recent work implicates that RICTOR amplification may define a unique subtype of NSCLC that can potentially be targeted by mTOR1/2 inhibitors. Additionally, previous evidence also suggest that the PI3K/AKT/mTOR pathway may be involved in immune regulation. Immunotherapy targeting PD-1/PD-L1 has significantly shifted the treatment landscape in NSCLC. PD-1/PD-L1 belongs to the B7 family of ligands and CD28 family of receptors, which play crucial roles in the regulation of T-cell responses. B7x, HHLA2 and B7-H3 are recently discovered B7 family ligands, which function as T-cell co-inhibitory molecules. These new immune checkpoints are widely expressed in NSCLC and in particular, these alternative immune checkpoints are commonly found in PD-L1 negative tumors. In the current study, we investigated the potential roles of the RICTOR/mTOR signaling on immune modulation, especially the regulation of immune checkpoints. Results: Using multiple RICTOR amplified NSCLC cells lines (H23, H1703 and H1734), we found that knockdown of RICTOR by siRNA or inducible shRNA resulted in decreased whole lysate and cell surface PD-L1 and HHLA-2 expression but not B7x or B7-H3. RICTOR knockdown can also downregulate IFN-gamma induced PD-L1 expression. Moreover, inducible RICTOR overexpression upregulates PD-L1 levels and also modestly HHLA2 expression. Our preliminary data also indicate that this regulation is likely occurring at a post-transcriptional level. In addition to potential modulation of immune checkpoint molecules, preliminary analysis of 14,698 NSCLC cases indicate that tumors with RICTOR amplification more often have higher levels of tumor mutation burden (TMB) than other lung cancers: the mean TMB was 14.9 mut/Mb in RICTOR-amplified vs 9.2 mut/Mb in non-amplified cases. TMB is a surrogate for increased number of expressed tumor neoantigens and is an important biomarker for response to immune checkpoint inhibitors. Conclusion: Our study provides new insight into the regulation of the tumor immune microenvironment by RICTOR/mTOR signaling. This may pave the way for designing or improving immunotherapeutic strategies in lung cancer by exploiting the novel immune escape mechanisms in these tumors. Citation Format: Mariam Alexander, Ni Fan, Xiaoxin Ren, Jeffrey Ross, Hao Wang, Feng Wang, Balazs Halmos, Roman Perez-Soler, Xingxing Zang, Haiying Cheng. RICTOR/mTOR signaling regulates novel immune checkpoints in non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1017.

Abstract 3387: Mechanisms driving clinical bone marrow-resident cancer cell quiescence

Abstract Despite widespread knowledge that bone marrow-resident breast cancer cells (BM-R BCs) affect disease progression and ultimately cause death, mechanisms regulating patient-derived BM-R BC quiescence remain poorly defined. This is caused by lack of biomarkers that can isolate quiescent but viable BM-R BCs devoid of cytokeratin and/or other epithelial markers. We developed and validated an experimental strategy to capture, isolate and characterize identify and isolate solitary BC cells from blood and BM of xenograft mice using a combination of human and BC-specific markers. These procedures allowed us to interrogate not only cytokeratin-positive but also unidentified cytokeratin-negative BC cell subsets. We hypothesized that subsets of patient-isolated circulating tumor cells (CTCs) will re-colonize various organs; and that underlying CTC signaling mechanisms will reflect respective transcriptomic signatures upon xeno-transplantation. Accordingly, we established an in vivo model of “latent” metastasis in CTC-derived xenografts (CDXs) by three sequential steps: a) isolation of CTC-enriched populations from metastatic BC patients, followed by their implantation in NSG mice; b) in vivo depletion of “non-tumor” human cells over an 8-month period; and c) capture and characterization of viable BC cells from blood and BM of CDX mice. These steps were implemented on patient samples from all primary BC subtypes along with validation of ex vivo CTC and BM-R BC gene signatures by whole genome transcriptomic arrays. First, three-way transcriptomic comparison between ex vivo BM-R BCs and CTCs vs de novo CTCs (obtained from GSE99394) revealed that ex vivo BMRBCs and CTCs clustered together and were largely similar (279 differentially regulated genes), implying that either the majority of ex vivo CTCs were shed from BM or that CTC transcriptomic signatures shed from other organs were similar to BM-shed CTCs. Second, comparative analyses of ex vivo BM-R BC vs de novo CTC gene signatures identified heightened mTORC2 vis-à-vis attenuated mTORC1 signaling as the most significant parameter of human BM-R BCs. Third, augmented levels of mTORC2-downstream targets were detected in quiescent (Ki67-/RBL2+) cells of paired metastatic vs primary BC tissues. Fourth, IHC analyses of CTC xenograft tissues revealed solitary BM and tissue-resident BC cells possessing high mTORC2. Lastly, shRNA knockdown of Rictor - the essential component of the mTORC2 complex - stimulated cell cycle progression and proliferation in vitro while depleting the BM-R BC population in vivo. Collectively, these findings suggest that the balance between mTORC2 vs mTORC1 interplays regulate the primordial stages of CTC quiescence. Deciphering mTORC2 signaling in CTC quiescence will proffer novel biomarkers for quiescent CTC detection and innovative therapeutic interventions in BC patients with undetectable metastasis. Citation Format: Debasish Boral, Monika Vishnoi, Haowen Liu, DARIO MARCHETTI. Mechanisms driving clinical bone marrow-resident cancer cell quiescence [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3387.

Overexpression of Rictor protein and Rictor-H. pylori interaction has impact on tumor progression and prognosis in patients with gastric cancer.

Growing evidence indicates that Rictor (Rapamycin-insensitive companion of mTOR) is overexpressed across several malignancies and associated with poor survival. However, only limited data indicate that Rictor plays a role in gastric cancer (GC). We sought to explore the prognostic value of Rictor in GC and present interaction analysis between Rictor expression and H. pylori status regarding their effects over the prognosis of GC patient. 250 GC tissues and 124 lymph node metastases were collected for the detection of Rictor by immunohistochemistry. Cox regression model was used to assess the association between Rictor expression and patient prognosis. Functional experiments were examined in transfected cells using Rictor siRNA. Additive and multiplicative interactions of Rictor and H. pylori were evaluated. In this study, the positive rate of Rictor was 51.6% (129/150) in GC tissues. Multivariate analyses showed that Rictor was independent unfavorable predictor for OS (HR = 1.554, 95% CI = 1.076-2.244, P = 0.019) and DFS (HR = 1.556, 95% CI = 1.081-2.240, P = 0.017). Patients with upregulated Rictor in the primary tumor and lymph node metastases had the worst prognosis. We observed significant additive and multiplicative interactions between Rictor expression and H. pylori status for OS and DFS (P < 0.05). Our in vitro experiment showed that knockdown of Rictor could suppress cell proliferation, induce apoptosis and inhibit tumor migration and invasion. Our results demonstrate that Rictor, acting as an oncogene, might be a potential prognostic biomarker and therapeutic target in GC. We suggest that Rictor expression and H. pylori status may be a prognostic marker in gastric cancer.

Molecular Interplay between Dormant Bone Marrow-Resident Cells (BMRCs) and CTCs in Breast Cancer.

Despite widespread knowledge that bone marrow-resident breast cancer cells (BMRCs) affect tumor progression, signaling mechanisms of BMRCs implicated in maintaining long-term dormancy have not been characterized. To overcome these hurdles, we developed a new experimental model of clinical dormancy employing patient-isolated Circulating Tumor Cells (de novo CTCs) and their injection in xenografts with subsequent tumor monitoring and CTC characterization (ex vivo CTCs). We hypothesized that significant distinctions exist between signaling pathways of bone marrow-homing vs metastasis-competent CTCs upon transplantation in xenografts. Comparative transcriptomic analyses of ex vivo vs de novo CTCs identified increased mTOR signaling—a critical pathway frequently dysregulated in breast cancer and implicated in cell survival and dormancy—with contrasting actions by its two complementary arms (mTORC2/mTORC1). Heightened mTORC2 downstream targets augmented quiescent CTCs (Ki67−/RBL2+ cells) in paired breast cancer tissues, along with high mTORC2 activity in solitary BMRCs and tissue-resident CTCs. Further, shRNA mediated the knockdown of RICTOR, an essential component of mTORC2, and augmented Ki67/PCNA biomarker expression and proliferation. Collectively, these findings suggest that the balance between mTORC1 vs mTORC2 signaling regulates CTC-associated mitotic and/or dormancy characteristics.

Regulation of Autophagy by Protein Kinase C-ε in Breast Cancer Cells.

Protein kinase C-ε (PKCε), an anti-apoptotic protein, plays critical roles in breast cancer development and progression. Although autophagy is an important survival mechanism, it is not known if PKCε regulates autophagy in breast cancer cells. We have shown that silencing of PKCε by siRNA inhibited basal and starvation-induced autophagy in T47D breast cancer cells as determined by the decrease in LC3-II, increase in p62, and decrease in autophagy puncta both in the presence and absence of bafilomycin A1. The mechanistic target of rapamycin (mTOR) associates with Raptor or Rictor to form complex-1 (mTORC1) or complex-2 (mTORC2), respectively. Knockdown of PKCε attenuated an increase in autophagy caused by the depletion of Raptor and Rictor. Overexpression of PKCε in MCF-7 cells caused activation of mTORC1 and an increase in LC3-I, LC3-II, and p62. The mTORC1 inhibitor rapamycin abolished the increase in LC3-I and p62. Knockdown of mTOR and Rictor or starvation enhanced autophagy in PKCε overexpressing cells. While overexpression of PKCε in MCF-7 cells inhibited apoptosis, it induced autophagy in response to tumor necrosis factor-α. However, inhibition of autophagy by Atg5 knockdown restored apoptosis in PKCε-overexpressing cells. Thus, PKCε promotes breast cancer cell survival not only by inhibiting apoptosis but also by inducing autophagy.

RICTOR amplification as a novel therapeutic target for lung cancer brain metastases.

3597 Background: Approximately 20% to 50% of patients with advanced lung cancer develop brain metastases, which are associated with debilitating neurologic impairment and a dismal prognosis. There have been very limited studies investigating the genomics of brain metastases in lung cancer. Methods: We comprehensively investigated the frequency of PI3K/AKT/RICTOR/mTOR pathway aberrations in primary and metastatic sites using an extensive database of 11845 cases of lung adenocarcinoma by NGS (FoundationOne). The potential roles of RICTOR amplification in the development of brain metastases were studied both in vitro and in vivo in orthotopic mouse models. Results: Compared to the primary tumor, PI3K/AKT/mTOR gene alterations were more frequent in metastatic sites, with particular enrichment noted in brain metastases. RICTOR amplification alone accounted for the observed higher frequency both in brain metastases (brain vs. primary: 9.73% vs 3.50%, P = 2.6E-14; brain vs. other mets: 9.73% vs. 7.3%, P = 0.03) and other metastatic sites (other mets vs. primary: 7.3% vs.3.5%, P = 10E-15), whereas the frequency of PTEN, AKT1, PK3CA or mTOR genetic alterations was not different in the primary tumor, brain and other metastatic sites. In vitro, inducible RICTOR knockdown in H23 lung cancer cells (parental line with RICTOR amplification) was associated with reduced cell migration and invasion, whereas upregulation of RICTOR in HCC827 lung cancer cells (parental line with normal RICTOR copy numbers) was associated with an increase of both processes. These results were confirmed with pharmacological inhibition using mTOR1/2 inhibitors with known CNS penetration. In vivo, both inducible ablation of RICTOR and the mTOR1/2 inhibitor TAK228 (Sapanisertinib) significantly inhibited lung cancer H23-R4-Luc tumor growth in the brain, including a number of near complete responses. Mechanistic studies suggest that RICTOR may regulate the brain metastasis process through AKT and CXCL12 chemokine-CXCR4 axis. Conclusions: RICTOR amplification is the first identified actionable target that is markedly enriched in brain metastases. Our study provides a strong rationale for the development of RICTOR-targeted therapeutic strategies for the treatment and/or prevention of these major causes of lung cancer morbidity and mortality.

Differential roles and activation of mammalian target of rapamycin complexes 1 and 2 during cell migration in prostate cancer cells

Mammalian target of rapamycin (mTOR) is a downstream substrate activated by PI3K/AKT pathway and it is essential for cell migration. It exists as two complexes: mTORC1 and mTORC2. mTORC1 is known to be regulated by active AKT, but the activation of mTORC2 is poorly understood. In this study, we investigated the roles and differential activation of the two mTOR complexes during cell migration in prostate cancer cells. We used small interfering RNA to silence the expression of Rac1 and the main components of mTOR complexes (regulatory associated protein of mTOR [RAPTOR] and rapamycin-insensitive companion of mTOR [RICTOR]) in LNCaP, DU145, and PC3 prostate cancer cell lines. We performed transwell migration assay to evaluate the migratory capability of the cells, and Western blot analysis to study the activation levels of mTOR complexes. Specific knockdown of RAPTOR and RICTOR caused a decrease of cell migration, suggesting their essential role in prostate cancer cell movement. Furthermore, epidermal growth factor (EGF) treatments induced the activation of both the mTOR complexes. Lack of Rac1 activity in prostate cancer cells blocked EGF-induced activation of mTORC2, but had no effect on mTORC1 activation. Furthermore, the overexpression of constitutively active Rac1 resulted in significant increase in cell migration and activation of mTORC2 in PC3 cells, but had no effect on mTORC1 activation. Active Rac1 was localized in the plasma membrane and was found to be in a protein complex, with RICTOR, but not RAPTOR. We suggest that EGF-induced activation of Rac1 causes the activation of mTORC2 via RICTOR. This mechanism plays a critical role in prostate cancer cell migration.

Rictor promotes cell migration and actin polymerization through regulating ABLIM1 phosphorylation in Hepatocellular Carcinoma.

As one of the most ominous malignancies, hepatocellular carcinoma (HCC) is frequently diagnosed at an advanced stage, owing to its aggressive invasion and metastatic spread. Emerging evidence has demonstrated that Rictor, as a unique component of the mTORC2, plays a role in cell migration, as it is dysregulated in various cancers, including HCC. However, the underlying molecular mechanism has not been well-characterized. Here, evaluation on a tissue-array panel and bioinformatics analysis revealed that Rictor is highly expressed in HCC tissues. Moreover, increased Rictor expression predicts poor survival of HCC patients. Rictor knockdown significantly suppressed cell migration and actin polymerization, thereby leading to decreased nuclear accumulation of MKL1 and subsequent inactivation of SRF/MKL1-dependent gene transcription, i.e. Arp3 and c-Fos. Mechanistically, we identified ABLIM1 as a previously unknown phosphorylation target of Rictor. Rictor interacts with ABLIM1 and regulates its serine phosphorylation in HCC cells. We generated ABLIM1 knockout cell lines of HCC, in which dominant negative mutations of Ser 214 and Ser 431 residues inhibited the ABLIM1-mediated actin polymerization and the MKL1 signaling pathway. Overall, ABLIM1 phosphorylation induced by Rictor plays an important role in controlling actin polymerization in HCC cells.

Combinatorial Inhibition of mTORC2 and Hsp90 Leads to a Distinctly Effective Therapeutic Strategy in Malignant Pheochromocytoma.

Malignant pheochromocytoma (mPCC) is an uncommon tumor with poor prognosis, and no effective therapeutic strategy exists as yet. Discovering new and effective therapeutic strategies to improve prognosis is an urgent need. To investigate whether a combinatorial inhibition of both mTORC2 and Hsp90 in PC12 cells could lead to a distinct anti-tumor effect in vitro and in vivo that was greater than the inhibition of mTORC2 or Hsp90 alone. Targeting mTORC2 was assessed by knockdown of Rictor using shRNA, and 17-AAG was used to inhibit Hsp90 function. Combinatorial inhibition of both mTORC2 and Hsp90 could lead to a distinct anti-tumor effect in vitro that was greater than the inhibition of mTORC2 or Hsp90 alone. Inhibiting Hsp90 specifically could inhibit tumor growth of sh-Rictor cells in vivo, suggesting that the combinatorial inhibition of both mTORC2 and Hsp90 could lead to a distinct anti-tumor effect in vivo. Western blotting has shown that both p-Akt Ser473 and p-Akt Thr450 showed significantly decreased expression after targeting mTORC2, while p-Akt Thr308 did not. However, all three different p-AKTs, including p-Akt Ser473, p-Akt Thr450 and p-Akt Thr308, showed a significantly decreased expression in combinatorial inhibition of both mTORC2 and Hsp90. Collectively, it revealed that combinatorial inhibition of mTORC2 and Hsp90 could destabilize the Akt signaling. Our results demonstrated that combinatorial inhibition of mTORC2 and Hsp90 could increase their anti-tumor effect and destabilize the Akt signaling in PC12 cells, suggesting a combinatorial inhibition of both mTORC2 and Hsp90 which might be an effective therapeutic strategy for mPCC.

Abstract 4748: RICTOR signaling modulates the activity of EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutated non-small cell lung cancer (NSCLC)

Abstract Background: Molecularly-targeted therapy has significantly improved the therapeutic efficiency and clinical outcome in a subgroup of NSCLC patients. However, drug resistance inevitably emerges, such as resistance to EGFR TKIs in EGFR-mutated NSCLC. RICTOR is a key component of the mTORC2 complex and plays important roles in cell survival, proliferation, migration and invasion. RICTOR has also been suggested to play a part in the regulation of drug resistance to HER2-targeted therapy in HER2-amplified breast cancer, a niche close to EGFR-targeted therapy. In our current study, we hypothesize that RICTOR modulates the activity of EGFR TKIs in EGFR-mutated NSCLC. Co-targeting RICTOR and EGFR signaling pathways could be a new therapeutic strategy for patients with EGFR-mutated lung cancer. Results: Genetically modified EGFR-mutated NSCLC cell lines with different RICTOR expression levels have been developed. RICTOR knockdown significantly increases the sensitivity to all three generations of TKI inhibitors (erlotinib, afatinib and osimertinib), whereas overexpression of RICTOR renders the cells more resistant to EGFR TKIs. In addition, RICTOR knockdown restores the sensitivity of erlotinib-resistant HCC827 cells due to AXL overexpression (IC50: 216µM vs. 0.256 µM with RICTOR knockdown, P&amp;lt;0.001). Pharmacological inhibition of the RICTOR signaling pathway with the mTOR1/2 inhibitor (sapanisertib) also improves EGFR TKI sensitivity. Taken together, RICTOR signaling modulates the efficacy of EGFR TKIs in the EGFR-mutated lung cancer models. Conclusion: Our study provides a new insight into the role of RICTOR signaling modulation in increasing efficacy of EGFR TKI therapy in EGFR-mutated NSCLC. The combination of mTOR1/2 inhibitor and EGFR inhibitor could be a potential novel strategy leading to better therapeutic efficacy. Note: This abstract was not presented at the meeting. Citation Format: Ni Fan, Yiyu Zou, Balazs Halmos, Roman Perez-Soler, Haiying Cheng. RICTOR signaling modulates the activity of EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutated non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4748.

Abstract 4305: AMPK-RUNX2-mTORC2 axis involved in EMT of MDA-MB-231 breast cancer cells

Abstract Metformin is one of the most widely used biguanides for the treatment of type 2 diabetes, whose role as a potent anti-cancer drug has emerged in recent times. Its function as an anti-tumor agent is attributed to its ability to inhibit mTORC1, which is a key player in regulating cell proliferation. However, the use of metformin as an anti-cancer drug is hampered by several limitations. Hence, in order to elucidate the detailed mechanistic properties of metformin in cell growth and metastasis, we investigated the effect of metformin on mTORC2 activity in MDA-MB-231 cells. We report a novel mechanism by which metformin can increase the activity of mTORC2 in a Runx2-dependent fashion, which requires AMPK mediated stabilization of RUNX2. Furthermore, we found that mTORC2 and RUNX2 have a feed forward relation whereby the knockdown of rictor, a key component in activating mTORC2, resulted in loss of RUNX2 through GSK3-β-mediated ubiquitination of RUNX2. Since both mTORC2 and RUNX2 have been found to be involved in activating the EMT, metformin treatment may contribute to this activation by stabilizing AMPK-RUNX2-mTORC2 axis. These results demonstrate that metformin-induced mechanisms can influence metastasis even if they inhibit cell growth, but the genetic heterogeneity of the malignancy may govern cell fate. Citation Format: Aramati Bindu Reddy, Meher B. Gayatri, Suresh Chava. AMPK-RUNX2-mTORC2 axis involved in EMT of MDA-MB-231 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4305.

Rictor regulates mitochondrial calcium signaling in mouse embryo stem cell-derived cardiomyocytes

To explore the expression, localization and regulatory effect on mitochondrial calcium signaling of Rictor in embryonic stem cell-derived cardiomyocytes (ESC-CMs). Classical embryonic stem cell cardiomyogenesis model was used for differentiation of mouse embryonic stem cells into cardiomyocytes. The location of Rictor in ESC-CMs was investigated by immunofluorescence and Western blot. The expression of Rictor in mouse embryonic stem cells was interfered with lentiviral technology, then the superposition of mitochondria and endoplasmic reticulum (ER) in ESC-CMs was detected with immunofluorescence method; the cellular ultrastructure of ESC-CMs was observed by transmission electron microscope; the mitochondrial calcium transients of ESC-CMs was detected by living cell workstation;immunoprecipitation was used to detect the interaction between 1,5,5-trisphosphate receptor (IP3 receptor, IP3R), glucose-regulated protein 75 (Grp75) and voltage-dependent anion channel 1 (VDAC1) in mitochondrial outer membrane; the expression of mitochondrial fusion protein (mitonusin-2, Mfn2) was detected by Western blot. Rictor was mainly localized in the endoplasmic reticulum and mitochondrial-endoplasmic reticulum membrane (MAM) in ESC-CMs. Immunofluorescence results showed that Rictor was highly overlapped with ER and mitochondria in ESC-CMs. After mitochondrial and ER were labeled with Mito-Tracker Red and ER-Tracker Green, it was demonstrated that the mitochondria of the myocardial cells in the Rictor group were scattered, and the superimposition rate of mitochondria and ER was lower than that of the negative control group (P<0.01). The MAM structures were decreased in ESC-CMs after knockdown of Rictor. The results of the living cell workstation showed that the amplitude of mitochondrial calcium transients by ATP stimulation in ESC-CMs was decreased after knockdown of Rictor (P<0.01). The results of co-immunoprecipitation showed that the interaction between IP3R, Grp75 and VDAC1 in the MAM structure of the cardiomyocytes in the Rictor group was significantly attenuated (P<0.01); the results of Western blot showed that the expression of Mfn2 protein was significantly decreased (P<0.01). Using lentiviral technology to interfere Rictor expression in mouse embryonic stem cells, the release of calcium from the endoplasmic reticulum to mitochondria in ESC-CMs decreases, which may be affected by reducing the interaction of IP3R, Grp75, VDAC1 and decreasing the expression of Mfn2, leading to the damage of MAM structure.

UVB-induced inactivation of manganese-containing superoxide dismutase promotes mitophagy via ROS-mediated mTORC2 pathway activation

Mitochondria are major sites of energy metabolism that influence numerous cellular events, including immunity and cancer development. Previously, we reported that the mitochondrion-specific antioxidant enzyme, manganese-containing superoxide dismutase (MnSOD), has dual roles in early- and late-carcinogenesis stages. However, how defective MnSOD impacts the chain of events that lead to cell transformation in pathologically normal epidermal cells that have been exposed to carcinogens is unknown. Here, we show that UVB radiation causes nitration and inactivation of MnSOD leading to mitochondrial injury and mitophagy. In keratinocytes, exposure to UVB radiation decreased mitochondrial oxidative phosphorylation, increased glycolysis and the expression of autophagy-related genes, and enhanced AKT Ser/Thr kinase (AKT) phosphorylation and cell growth. Interestingly, UVB initiated a prosurvival mitophagy response by mitochondria-mediated reactive oxygen species (ROS) signaling via the mammalian target of the mTOR complex 2 (mTORC2) pathway. Knockdown of rictor but not raptor abrogated UVB-induced mitophagy responses. Furthermore, fractionation and proximity-ligation assays reveal that ROS-mediated mTOC2 activation in mitochondria is necessary for UVB-induced mitophagy. Importantly, pretreatment with the MnSOD mimic MnTnBuOE-2-PyP5+ (MnP) attenuates mTORC2 activation and suppresses UVB-induced mitophagy. UVB radiation exposure also increased cell growth as assessed by soft-agar colony survival and cell growth assays, and pretreatment with MnP or the known autophagy inhibitor 3-methyladenine abrogated UVB-induced cell growth. These results indicate that MnSOD is a major redox regulator that maintains mitochondrial health and show that UVB-mediated MnSOD inactivation promotes mitophagy and thereby prevents accumulation of damaged mitochondria.

Hippocampus-specific Rictor knockdown inhibited 17β-estradiol induced neuronal plasticity and spatial memory improvement in ovariectomized mice.

Estrogens have been shown to play profound roles in the regulation of the structure and function of the hippocampus; however, the underlying mechanism is not clear. Previous studies have shown that when Rictor, the core component of the mammalian target of the rapamycin complex 2 (mTORC2), was deleted, hippocampal actin polymerization was reduced and long-term memory was seriously impaired. Although hippocampal Rictor could be regulated by estrogen receptor agonists/antagonists, whether Rictor could directly mediate estrogenic regulation of neuronal plasticity, spatial learning and memory remains unclear. In this study, we first examined the regulation of hippocampal Rictor and P-AKTser473 (P-AKT) by E2, then we used Rictor-specific dsRNA (shRictor) injected into the hippocampi of E2-treated ovariectomized (OVX) mice or into cultured cells. The results showed that both Rictor and P-AKT could be regulated by E2. OVX induced actin depolymerization, decreases in CA1 spine density and synapse density as well as changes in synaptic proteins were reversed by E2 replacement. However, these E2-mediated effects were significantly blocked by shRictor treatment. Similar results were also demonstrated by in vitro cell culture studies using E2 and/or shRictor. Importantly, we found that E2 replacement induced improvements in learning and memory impairment seen in OVX mice were significantly blocked by shRictor. Taken together, the current studies provided the first direct evidence for the important role of Rictor in estrogenic action on the hippocampus, indicating that it may be a therapeutic target for the treatment of E2-related, hippocampus-dependent cognitive dysfunction.

Critical Role of mTORC2-Akt Signaling in TGF-β1-Induced Myofibroblast Differentiation of Human Pterygium Fibroblasts

Profibrotic activation is essential for pterygium development. In this study, we investigated the role of the mechanistic target of rapamycin (mTOR) in regulating TGF-β1-induced myofibroblastic responses in human pterygium fibroblasts (HPFs) and elucidated the relative contributions of mTOR signaling components. HPFs were pretreated with the mTOR inhibitors rapamycin and Torin2, and TGF-β1-induced expression of profibrotic markers, including α-smooth muscle actin (α-SMA) and fibronectin, was evaluated. RNA interference-based approaches targeting raptor and rictor, regulatory subunits of mTOR complex 1 (mTORC1) and 2 (mTORC2), respectively, were used to determine the impact of each mTOR complex on HPFs. The contractile phenotype of HPFs was assessed by a collagen gel contraction assay. The mTOR active-site inhibitor Torin2, which suppresses both mTORC1 and mTORC2 activity in HPFs, inhibited TGF-β1-induced expression of α-SMA and fibronectin. The allosteric inhibitor rapamycin only partially suppressed mTORC1 activity and exhibited a minimal effect on the induction of profibrotic markers. The induction of α-SMA and fibronectin in HPFs was abrogated by RNA interference-mediated knockdown of rictor but was only moderately affected by raptor knockdown. Akt inhibition mimicked the effect of Torin2 and rictor knockdown on myofibroblast differentiation of HPFs. mTOR inhibition potently reduced the contractile ability of HPFs in collagen gel contraction assays. This study found that mTOR signaling promoted profibrotic activation of HPFs and confirmed the importance of the mTORC2-Akt axis in TGF-β1-induced myofibroblast differentiation. Therefore, our study may open up new avenues for the development of novel therapeutic strategies involving targeting of mTOR signaling to treat pterygium.

Legionella pneumophila infection-mediated regulation of RICTOR via miR-218 in U937 macrophage cells

BackgroundInhalation of aerosolized Legionella pneumophila, a Gram-negative bacterium, can cause severe pneumonia. During infection, L. pneumophila replicates intracellularly in macrophages. The involvement of host microRNAs (miRNAs) in L. pneumophila infection is not fully understood. MethodsThe human macrophage-like cell line U937 was infected with L. pneumophila. The levels of miRNA and messenger RNA (mRNA) were measured using reverse transcriptase polymerase chain reaction. Release of lactate dehydrogenase was used to evaluate cytotoxicity. The expression of RICTOR and related proteins was examined by western blotting of cell lysates. ResultsL. pneumophila infection upregulated the expression of miR-218 and the host genes SLIT2 and SLIT3 in U937 cells. The expression of RICTOR, a component of the mechanistic target of rapamycin complex 2 (mTORC2), decreased during L. pneumophila infection. RICTOR protein expression was inhibited by the overexpression of miR-218, whereas knockdown of miR-218 restored the downregulation of RICTOR by L. pneumophila. L. pneumophila infection induced the expression of the proinflammatory cytokines IL-6 and TNF-alpha, which was modulated by knockdown of miR-218 or RICTOR. ConclusionsOur study revealed the involvement of miR-218 in regulating the inflammatory response of macrophages against L. pneumophila infection. These findings suggest potential novel roles for miR-218 and RICTOR as therapeutic targets of L. pneumophila infection.

181 - Manganese superoxide dismutase regulates mTORC2 mediated UVB-induced mitophagy

Mitochondria are major sites of energy metabolism that control numerous cellular events including immunity and cancer development. Previously, we reported the dual roles of MnSOD in early- and late- carcinogenesis stages. However, how MnSOD regulates the chain of events in the pathologically normal epithelial cells that have been exposed to carcinogens is unknown. Here, we show that UVB radiation causes nitration and inactivation of MnSOD activity in vitro and in vivo leading to initiation of mitochondrial injury and mitophagy. In primary human keratinocytes, exposure to UVB radiation decreases mitochondrial oxidative phosphorylation, increases glycolysis, increases the expression of autophagy-related genes, and enhances AKT phosphorylation and cell proliferation. Interestingly, UVB initiates a prosurvival mitophagy response by mitochondria-mediated reactive oxygen species (ROS) signaling via the mammalian target of the rapamycin complex 2 (mTORC2) pathway. Exposure to UVB increases rictor but not raptor expression, and overexpression of rictor increases mitophagy. Conversely, knock-down of rictor but not raptor abrogates UVB-induced mitophagy responses. Furthermore, fractionation and proximity-ligation assays reveal that mitochondrial ROS- mediated mTOC2 translocation to mitochondria is necessary for UVB-induced mitophagy. Importantly, pretreatment with MnTnBuOE-2-PyP5+ (MnP), a mitochondrial superoxide dismutase mimetic, attenuates mTORC2 activation and suppresses UVB-induced mitophagy. Exposure to UVB radiation increases cell transformation as assessed by soft-agar colony survival assay and pretreatment with MnP or 3-MA, a known autophagy inhibitor, abrogates UVB-induced cell transformation. These results demonstrate for the first time that MnSOD is a redox sensitive protein that maintains mitochondrial health and prevents UVB-induced mitophagy and cell transformation by regulating the activity of the mTORC2 complex.

Stress-induced modulation of volume-regulated anions channels in human alveolar carcinoma cells.

Shift in the cellular homeostasis of the organic osmolyte taurine has been associated with dysregulation of the volume‐regulated anion channel (VRAC) complex, which comprises leucine‐rich repeat‐containing family 8 members (LRRC8A‐E). Using SDS‐PAGE, western blotting, qRT‐PCR, and tracer technique ([3H]taurine) we demonstrate that reactive oxygen species (ROS) and the cell growth‐associated kinases Akt/mTOR, play a role in the regulation of VRAC in human alveolar cancer (A549) cells. LRRC8A is indispensable for VRAC activity and long‐term exposure to hypoosmotic challenges and/or ROS impairs VRAC activity, not through reduction in total LRRC8A expression or LRRC8A availability in the plasma membrane, but through oxidation/inactivation of kinases/phosphatases that control VRAC activity once it has been instigated. Pursuing Akt signaling via the serine/threonine kinase mTOR, using mTORC1 inhibition (rapamycin) and mTORC2 obstruction (Rictor knockdown), we demonstrate that interference with the PI3K‐mTORC2‐Akt signaling‐axes obstructs stress‐induced taurine release. Furthermore, we show that an increased LRRC8A expression, following exposure to cisplatin, ROS, phosphatase/lipoxygenase inhibitors, and antagonist of CysLT1‐receptors, correlates an increased activation of the proapoptotic transcription factor p53. It is suggested that an increase in LRRC8A protein expression could be taken as an indicator for cell stress and limitation in VRAC activity.