mTORC1 Complex Research Articles

Regulatory-Associated Protein of mTOR-Mediated Signaling: A Nexus Between Tumorigenesis and Disease

RAPTOR (regulatory-associated protein of mTOR) is a pivotal component of the mammalian target of rapamycin complex 1 (mTORC1), playing a central role in regulating cell growth, metabolism and stress responses. As a scaffold protein, RAPTOR recruits key substrates such as eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and ribosomal protein S6 kinase (S6K), facilitating their phosphorylation by mTORC1, which in turn drives protein synthesis, lipid metabolism and cellular proliferation. Its regulatory function becomes especially crucial under conditions of nutrient deprivation or stress, where it enhances the stability of the mTORC1 complex, allowing cells to adapt to fluctuating environmental cues. The hyperactivation of mTORC1, largely mediated by RAPTOR, is frequently observed in various cancers, contributing to uncontrolled cell proliferation and tumorigenesis. Moreover, RAPTOR’s modulation of immune responses and metabolic pathways extends its influence beyond oncogenesis, impacting inflammatory diseases and metabolic disorders. This review meticulously elucidates RAPTOR’s structure, post-translational modifications as well as its indispensable role within the mTORC1 complex, emphasizing its regulatory functions in cellular growth, metabolic adaptation, immune response and disease pathology including oncogenesis. Furthermore, it explores emergent therapeutic avenues targeting RAPTOR-mediated mTORC1 signaling, underscoring their potential to revolutionize cancer treatment and the management of related pathophysiological conditions.

MTORC1 and mTORC2 Levels in Patients With Psoriasis

Introduction: In recent years, the role of the mammalian target of rapamycin (mTOR) pathway, which is one of the intracellular signaling pathways and known as the main control pathway of metabolism, in the pathogenesis of psoriasis has been emphasized. Objectives: We sought to investigate the importance of the mTOR pathway in the pathogenesis of psoriasis. Methods: Forty patients with psoriasis and 40 healthy volunteers were included in this case-control study. Serum fasting mTORC1 and mTORC2 levels of the study groups were examined by enzyme-linked immunosorbent assay. Results: Serum mTORC1 and mTORC2 levels were significantly lower in patients with psoriasis than controls (p= 0.001). A positive correlation was found between serum mTORC1 and serum mTORC2 levels in patients with psoriasis (p=0,001, r=0,826). Conclusion: The lower serum levels of mTORC1 and mTORC2 complexes which are active signalling molecules in the cell, were found to be lower in patient with psoriasis, suggesting that it may be an indicator of increased intracellular activation of these molecules. Our opinion that agents that can effectively inhibit both mTOR complexes may be more effective in the treatment of psoriasis.

Veratridine, a plant-derived alkaloid, suppresses the hyperactive Rictor-mTORC2 pathway: a new targeted therapy for primary and metastatic colorectal cancer.

Despite considerable advances to improve colorectal cancer (CRC) survival over the last decade, therapeutic challenges remain due to the rapid metastatic dissemination of primary tumors and screening limitations. Meanwhile, the rise of CRC in younger adults (Early-onset CRC), commonly diagnosed with a metastatic form of the disease, shows the pressing need to develop more effective targeted therapies to decrease the high mortality rates associated with metastatic disease. Hyperactivation of the Rictor-mTORC2-AKT signaling pathway drives key metastatic players in diverse malignant tumors, including early- and late-onset colorectal cancer. Selective mTORC2 inhibitors are becoming a potential treatment strategy for CRC due to the therapeutic limitations of mTORC1 inhibitors. Veratridine (VTD), a lipid-soluble alkaloid extracted from Liliaceae plants, can transcriptionally increase UBXN2A, which induces 26S proteasomal degradation of the Rictor protein, a key member in the mTORC2 complex. Destabilization of Rictor protein by VTD decreases Akt phosphorylation on Ser 473 , which is responsible for metastatic signaling downstream of the mTORC2 pathway in diverse malignant tumors. VTD decreases the population of metastatic colon cancer stem cells and functions as an angiogenesis inhibitor. VTD effectively reduces the spheroid growth rate and restricts cell migration. Live cell migration and invasion assays alongside biomechanical-force-based experiments revealed that VTD suppresses colon cancer cell invasiveness and the ensuing risk of tumor metastasis. A CRC mouse model that mimics the natural stages of human sporadic CRC revealed that VTD treatment significantly decreases tumor growth in a UBXN2A-dependent manner. This study showed a novel mechanistic connection between a ubiquitin-like protein and mTORC2-dependent migration and invasion in CRC tumors. This study revealed the therapeutic benefit of selective inhibition of Rictor in CRC, particularly in tumors with a hyperactive Rictor-mTORC2 signaling pathway. Finally, this study opened a new platform for repurposing VTD, a supplemental anti-hypertension molecule, into an effective targeted therapy in CRC tumors.

Reciprocal regulation of mTORC1 signaling and ribosomal biosynthesis determines cell cycle progression in activated T cells.

Ribosomal biosynthesis in nucleoli is an energy-demanding process driven by all RNA polymerases and hundreds of auxiliary proteins. We investigated how this process is regulated in activated T lymphocytes by T cell receptor (TCR) signals and the multiprotein complexes mTORC1 and mTORC2, both of which contain the kinase mTOR. Deficiency in mTORC1 slowed the proliferation of T cells, with further delays in each consecutive division, an effect not seen with deficiency in mTORC2. mTORC1 signaling was stimulated by components of conventional TCR signaling, and, reciprocally, TCR sensitivity was decreased by mTORC1 inhibition. The substantial increase in the amount of RNA per cell induced by TCR activation was reduced by 50% by deficiency in mTORC1, but not in mTORC2 or in S6 kinases 1 and 2, which are activated downstream of mTORC1. RNA-seq data showed that mTORC1 deficiency reduced the abundance of all RNA biotypes, although rRNA processing was largely intact in activated T cells. Imaging cytometry with FISH probes for nascent pre-rRNA revealed that deletion of mTORC1, but not that of mTORC2, reduced the number and expansion of nucleolar sites of active transcription. Protein translation was consequently decreased by 50% in the absence of mTORC1. Inhibiting RNA polymerase I blocked not only proliferation but also mTORC1 signaling. Our data show that TCR signaling, mTORC1 activity, and ribosomal biosynthesis in the nucleolus regulate each other during biomass production in clonally expanding T cells.

Placental Transport of Amino Acids in Rats with Methionine-Induced Hyperhomocysteinemia.

Maternal hyperhomocysteinemia (HHcy) is a risk factor for intrauterine growth restriction presumably caused by a decrease in the placental transport of nutrients. We investigated the effect of experimental HHcy induced by daily methionine administration to pregnant rats on the free amino acid levels in the maternal and fetal blood, as well as on morphological and biochemical parameters associated with the amino acid transport through the placenta. HHcy caused an increase in the levels of most free amino acids in the maternal blood on gestational day 20, while the levels of some amino acids in the fetal blood were decreased. In rats with HHcy, the maternal sinusoids in the placental labyrinth were narrowed, which was accompanied by aggregation of red blood cells. We also observed an increase in the neutral amino acid transporters (LAT1, SNAT2) protein levels and activation of 4E-BP1, a downstream effector of mTORC1 complex, in the labyrinth zone. Maternal HHcy affected the placental barrier permeability, as evidenced by intensification of the mother-to-fetus transfer of Evans Blue dye. The imbalance in the free amino acid levels in the maternal and fetal blood in HHcy may be due to the competition of homocysteine with other amino acids for common transporters, as well as a decrease in the area of exchange zone between maternal and fetal circulations in the placental labyrinth. Upregulation of the neutral amino acid transporter expression in the labyrinth zone may be a compensatory response to an insufficient intrauterine amino acid supply and fetal growth restriction.

Assembly of mTORC3 Involves Binding of ETV7 to Two Separate Sequences in the mTOR Kinase Domain.

mTOR plays a crucial role in cell growth by controlling ribosome biogenesis, metabolism, autophagy, mRNA translation, and cytoskeleton organization. It is a serine/threonine kinase that is part of two distinct extensively described protein complexes, mTORC1 and mTORC2. We have identified a rapamycin-resistant mTOR complex, called mTORC3, which is different from the canonical mTORC1 and mTORC2 complexes in that it does not contain the Raptor, Rictor, or mLST8 mTORC1/2 components. mTORC3 phosphorylates mTORC1 and mTORC2 targets and contains the ETS transcription factor ETV7, which binds to mTOR and is essential for mTORC3 assembly in the cytoplasm. Tumor cells that assemble mTORC3 have a proliferative advantage and become resistant to rapamycin, indicating that inhibiting mTORC3 may have a therapeutic impact on cancer. Here, we investigate which domains or amino acid residues of ETV7 and mTOR are involved in their mutual binding. We found that the mTOR FRB and LBE sequences in the kinase domain interact with the pointed (PNT) and ETS domains of ETV7, respectively. We also found that forced expression of the mTOR FRB domain in the mTORC3-expressing, rapamycin-resistant cell line Karpas-299 out-competes mTOR for ETV7 binding and renders these cells rapamycin-sensitive in vivo. Our data provide useful information for the development of molecules that prevent the assembly of mTORC3, which may have therapeutic value in the treatment of mTORC3-positive cancer.

The mTOR pathway controls phosphorylation of BRAF at T401

BRAF serves as a gatekeeper of the RAS/RAF/MEK/ERK pathway, which plays a crucial role in homeostasis. Since aberrant signalling of this axis contributes to cancer and other diseases, it is tightly regulated by crosstalk with the PI3K/AKT/mTOR pathway and ERK mediated feedback loops. For example, ERK limits BRAF signalling through phosphorylation of multiple residues. One of these, T401, is widely considered as an ERK substrate following acute pathway activation by growth factors. Here, we demonstrate that prominent T401 phosphorylation (pT401) of endogenous BRAF is already observed in the absence of acute stimulation in various cell lines of murine and human origin. Importantly, the BRAF/RAF1 inhibitor naporafenib, the MEK inhibitor trametinib and the ERK inhibitor ulixertinib failed to reduce pT401 levels in these settings, supporting an alternative ERK-independent pathway to T401 phosphorylation. In contrast, the mTOR inhibitor torin1 and the dual-specific PI3K/mTOR inhibitor dactolisib significantly suppressed pT401 levels in all investigated cell types, in both a time and concentration dependent manner. Conversely, genetic mTOR pathway activation by oncogenic RHEB (Q64L) and mTOR (S2215Y and R2505P) mutants substantially increased pT401, an effect that was reverted by dactolisib and torin1 but not by trametinib. We also show that shRNAmir mediated depletion of the mTORC1 complex subunit Raptor significantly enhanced the suppression of T401 phosphorylation by a low torin1 dose, while knockdown of the mTORC2 complex subunit Rictor was less effective. Using mass spectrometry, we provide further evidence that torin1 suppresses the phosphorylation of T401, S405 and S409 but not of other important regulatory phosphorylation sites such as S446, S729 and S750. In summary, our data identify the mTOR axis and its inhibitors of (pre)clinical relevance as novel modulators of BRAF phosphorylation at T401.

The Autophagic and Apoptotic Death of Forebrain Neurons of Rats with Global Brain Ischemia Is Diminished by the Intranasal Administration of Insulin: Possible Mechanism of Its Action.

Insulin is a promising neuroprotector. To better understand the mechanism of insulin action, it was important to show its ability to diminish autophagic neuronal death in animals with brain ischemic and reperfusion injury. In forebrain ischemia and reperfusion, the number of live neurons in the hippocampal CA1 region and frontal cortex of rats decreased to a large extent. Intracerebroventricular administration of the autophagy and apoptosis inhibitors to ischemic rats significantly increased the number of live neurons and showed that the main part of neurons died from autophagy and apoptosis. Intranasal administration of 0.5 IU of insulin per rat (before ischemia and daily during reperfusion) increased the number of live neurons in the hippocampal CA1 region and frontal brain cortex. In addition, insulin significantly diminished the level of autophagic marker LC3B-II in these forebrain regions, which markedly increased during ischemia and reperfusion. Our studies demonstrated for the first time the ability of insulin to decrease autophagic neuronal death, caused by brain ischemia and reperfusion. Insulin administered intranasally activated the Akt-kinase (activating the mTORC1 complex, which inhibits autophagy) and inhibited the AMP-activated protein kinase (which activates autophagy) in the hippocampus and frontal cortex of rats with brain ischemia and reperfusion.

Coordinated Regulation of Renal Glucose Reabsorption and Gluconeogenesis by mTORC2 and Potassium.

The kidney proximal tubule is uniquely responsible for reabsorption of filtered glucose and gluconeogenesis (GNG). Insulin stimulates glucose transport and suppresses GNG in the proximal tubule, however, the signaling mechanisms and coordinated regulation of these processes remain poorly understood. The kinase complex mTORC2 is critical for regulation of growth, metabolism, solute transport, and electrolyte homeostasis in response to a wide array of inputs. Here we examined its role in the regulation of renal glucose reabsorption and GNG. Rictor, an essential component of mTORC2, was knocked out using the Pax8-LC1 system to generate inducible tubule specific Rictor knockout (TRKO) mice. These animals were subjected to fasting, refeeding, and variation in dietary K + . Metabolic parameters including glucose homeostasis and renal function were assessed in balance cages. Kidneys and livers were also harvested for molecular analysis of gluconeogenic enzymes, mTORC2-regulated targets, and plasma membrane glucose transporters. On a normal chow diet, TRKO mice had marked glycosuria despite indistinguishable blood glucose relative to WT controls. Kidney plasma membrane showed lower SGLT2 and SGLT1 in the fed state, supporting reduced renal glucose reabsorption. Additional metabolic testing provided evidence for renal insulin resistance with elevated fasting insulin, impaired pyruvate tolerance, elevated hemoglobin A1c, and increased renal gluconeogenic enzymes in the fasted and fed states. These effects were correlated with reduced downstream phosphorylation of Akt and the transcription factor FOXO4, identifying a novel role of FOXO4 in the kidney. Interestingly, high dietary K + prevented glycosuria and excessive GNG in TRKO mice, despite persistent reduction in mTORC2 substrate phosphorylation. Renal tubule mTORC2 is critical for coordinated regulation of sodium-glucose cotransport by SGLT2 and SGLT1 as well as renal GNG. Dietary K + promotes glucose reabsorption and suppresses GNG independently of insulin signaling and mTORC2, potentially providing an alternative signaling mechanism in states of insulin resistance. The kidney contributes to regulation of blood glucose through reabsorption of filtered glucose and gluconeogenesis. This study shows that mTORC2 and dietary potassium coordinate the regulation of sodium-glucose cotransport and glucose production in the kidney via independent mechanisms. New insights into the regulation of these processes in the kidney offer promising implications for diabetes mellitus management and treatment.

Role of PIM Kinase Inhibitor in the Treatment of Alzheimer's Disease.

Alzheimer's disease (AD), a neurodegenerative disorder, is the most prevalent form of senile dementia, causing progressive deterioration of cognition, behavior, and rational skills. Neuropathologically, AD is characterized by two hallmark proteinaceous aggregates: amyloid beta (Aβ) plaques and neurofibrillary tangles (NFTs) formed of hyperphosphorylated tau. A significant study has been done to understand how Aβ and/or tau accumulation can alter signaling pathways that affect neuronal function. A conserved protein kinase known as the mammalian target of rapamycin (mTOR) is essential for maintaining the proper balance between protein synthesis and degradation. Overwhelming evidence shows mTOR signaling's primary role in age-dependent cognitive decline and the pathogenesis of AD. Postmortem human AD brains consistently show an upregulation of mTOR signaling. Confocal microscopy findings demonstrated a direct connection between mTOR and intraneuronal Aβ42 through molecular processes of PRAS40 phosphorylation. By attaching to the mTORC1 complex, PRAS40 inhibits the activity of mTOR. Furthermore, inhibiting PRAS40 phosphorylation can stop the Aβ-mediated increase in mTOR activity, indicating that the accumulation of Aβ may aid in PRAS40 phosphorylation. Physiologically, PRAS40 is phosphorylated by PIM1 which is a serine/threonine kinase of proto-oncogene PIM kinase family. Pharmacological inhibition of PIM1 activity prevents the Aβ-induced mTOR hyperactivity in vivo by blocking PRAS40 phosphorylation and restores cognitive impairments by enhancing proteasome function. Recently identified small-molecule PIM1 inhibitors have been developed as potential therapeutic to reduce AD-neuropathology. This comprehensive study aims to address the activity of PIM1 inhibitor that has been tested for the treatment of AD, in addition to the pharmacological and structural aspects of PIM1.

Macula densa mTORC1 is an important regulator of cellular and aging-dependent kidney functions

Macula densa (MD) cells are major regulators of nephron, kidney, and whole body functions, including the control of renal blood flow, glomerular filtration rate (GFR), renin release and thus the maintenance of body fluid and electrolyte balance. Our recent studies identified the high rate of MD protein synthesis regulated by the mTORC1 complex of the mechanistic target of rapamycin (mTOR) as a novel component of MD cell and glomerular hemodynamic regulatory functions. Raptor is an essential player in mTORC1’s key roles to regulate cell energy homeostasis, growth, protein synthesis, mitochondrial function, apoptosis, and autophagy via phosphorylation of substrates including ribosomal protein S6 kinase (S6K) and eukaryotic translation-initiation factor 4E-binding proteins (4E-BPs). mTORC1 signaling controls the aging process, and deficient mTORC1 activity extends health and lifespan. Therefore, we hypothesized that MD cell-specific mTOR loss of function (lof) prevents aging-related kidney function decline. The present study aimed to demonstrate the key importance of MD cell mTOR activity in renal physiology and aging by characterizing the phenotype of a newly generated genetic MD-mTOR lof mouse model. MD-mTOR lof mice were generated by crossing Nos1-CreERT2 and Raptor floxed mice. Tamoxifen induction (at 3 weeks of age) of Nos1-CreERT2+/- Raptor−/− mice resulted in conditional and inducible Raptor deletion in cells of the Nos1 lineage, which in the kidney is entirely specific to MD cells. Kidney structure/function phenotyping was performed using intravital multiphoton microscopy (MPM), the Medibeacon transdermal GFR measurement system, and tissue harvest, immunohistochemistry, and the OPP incorporation assay of protein synthesis. High and MD-specific Raptor, pS6K, and p4EBP1 immunofluorescence labeling was observed in WT kidney sections. The immunolabeling of these mTOR component and targets were absent specifically in MD cells in MD-mTOR lof mouse kidneys, validating the MD cell-specific genetic approach. The intensity of incorporated OPP labeling was highest in MD cells among all renal cell types in WT. In contrast, MD OPP labeling was significantly reduced in MD-mTOR lof mice indicating diminished, mTORC1-dependent global MD cell protein synthesis. Importantly, in 15-month old aged mice the GFR was significantly higher (preserved) in MD-mTOR lof (1794±197 uL/min/100g BW) compared to WT (1117±61) and MD-mTOR gof (929±61) mice (n=6-18 mice in each group). Altogether, these studies identified mTORC1 as an essential regulator of MD cell biology, and the importance of MD cells in aging-related kidney function decline. MD cells may be important new targets in future anti-aging therapies. DK064234, DK123564, DK135290. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Malvidin attenuates trauma-induced heterotopic ossification of tendon in rats by targeting Rheb for degradation via the ubiquitin-proteasome pathway.

The pathogenesis of trauma-induced heterotopic ossification (HO) in the tendon remains unclear, posing a challenging hurdle in treatment. Recognizing inflammation as the root cause of HO, anti-inflammatory agents hold promise for its management. Malvidin (MA), possessing anti-inflammatory properties, emerges as a potential agent to impede HO progression. This study aimed to investigate the effect of MA in treating trauma-induced HO and unravel its underlying mechanisms. Herein, the effectiveness of MA in preventing HO formation was assessed through local injection in a rat model. The potential mechanism underlying MA's treatment was investigated in the tendon-resident progenitor cells of tendon-derived stem cells (TDSCs), exploring its pathway in HO formation. The findings demonstrated that MA effectively hindered the osteogenic differentiation of TDSCs by inhibiting the mTORC1 signalling pathway, consequently impeding the progression of trauma-induced HO of Achilles tendon in rats. Specifically, MA facilitated the degradation of Rheb through the K48-linked ubiquitination-proteasome pathway by modulating USP4 and intercepted the interaction between Rheb and the mTORC1 complex, thus inhibiting the mTORC1 signalling pathway. Hence, MA presents itself as a promising candidate for treating trauma-induced HO in the Achilles tendon, acting by targeting Rheb for degradation through the ubiquitin-proteasome pathway.

P4HA2 activates mTOR via hydroxylation and targeting P4HA2-mTOR inhibits lung adenocarcinoma cell growth

,Mammalian target of rapamycin (mTOR) kinase functions as a central regulator of cell growth and metabolism, and its complexes mTORC1 and mTORC2 phosphorylate distinct substrates. Dysregulation of mTOR signaling is commonly implicated in human diseases, including cancer. Despite three decades of active research in mTOR, much remains to be determined. Here, we demonstrate that prolyl 4-hydroxylase alpha-2 (P4HA2) binds directly to mTOR and hydroxylates one highly conserved proline 2341 (P2341) within a kinase domain of mTOR, thereby activating mTOR kinase and downstream effector proteins (e.g. S6K and AKT). Moreover, the hydroxylation of P2341 strengthens mTOR stability and allows mTOR to accurately recognize its substrates such as S6K and AKT. The growth of lung adenocarcinoma cells overexpressing mTORP2341A is significantly reduced when compared with that of cells overexpressing mTORWT. Interestingly, in vivo cell growth assays show that targeting P4HA2-mTOR significantly suppresses lung adenocarcinoma cell growth. In summary, our study reveals an undiscovered hydroxylation-regulatory mechanism by which P4HA2 directly activates mTOR kinase, providing insights for therapeutically targeting mTOR kinase-driven cancers.

Rapamycin improves the survival of epilepsy model cells by blocking phosphorylation of mTOR base on computer simulations and cellular experiments

PurposeEpilepsy is a chronic brain dysfunction characterized by recurrent epileptic seizures. Rapamycin is a naturally occurring macrolide from Streptomyces hygroscopicus, and rapamycin may provide a protective effect on the nervous system by affecting mTOR. Therefore, we investigated the pharmacologic mechanism of rapamycin treating epilepsy through bioinformatics analysis, cellular experiments and supercomputer simulation. MethodsBioinformatics analysis was used to analyze targets of rapamycin treating epilepsy. We established epilepsy cell model by HT22 cells. RT-qPCR, WB and IF were used to verify the effects of rapamycin on mTOR at gene level and protein level. Computer simulations were used to model and evaluate the stability of rapamycin binding to mTOR protein. ResultsBioinformatics indicated mTOR played an essential role in signaling pathways of cell growth and cell metabolism. Cellular experiments showed that rapamycin could promote cell survival, and rapamycin did not have an effect on mRNA expression of mTOR. However, rapamycin was able to significantly inhibit the phosphorylation of mTOR at protein level. Computer simulations indicated that rapamycin was involved in the treatment of epilepsy through regulating phosphorylation of mTOR at protein level. ConclusionWe found that rapamycin was capable of promoting the survival of epilepsy cells by inhibiting the phosphorylation of mTOR at protein level, and rapamycin did not have an effect on mRNA expression of mTOR. In addition to the traditional study that rapamycin affects mTORC1 complex by acting on FKBP12, this study found rapamycin could also directly block the phosphorylation of mTOR, therefore affecting the assembly of mTORC1 complex and mTOR signaling pathway.

Abstract 4319: HSF1 inhibition induces pancreatic ductal adenocarcinoma autophagy through JNK

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related mortality, with a 5-year survival rate of 12%. Chemoresistance due to autophagy, a natural process of proteotoxic stress response, is the most common chemotherapy challenge for pancreatic cancer. Autophagy, which maintains cellular homeostasis by removing damaged or unnecessary proteins and organelles, is a self-degradation process regulated by the mammalian target of rapamycin complex (mTORC1). Excessive levels of autophagy can be detrimental and lead to cell death. Nevertheless, this dual function of autophagy in regulating pancreatic cancer remains poorly understood. It has been reported that heat shock factor 1 (HSF1), a critical pro-oncogenic transcription factor in the proteotoxic stress response, inhibits c-Jun N-terminal kinase 1/2 (JNK1/2) signaling pathway to maintain mTORC1 activity and control cell size. HSF1 is highly expressed in pancreatic tumors, and inhibition of HSF1 via small molecules enhances the chemotherapy drug effect on PDAC programmed cell death. However, the PDAC autophagy response in HSF1 inhibition is currently not fully understood. Objective: The objective of this study is to investigate the molecular mechanism of HSF1 inhibition in PDAC autophagy. Methods and Results: In human PDAC cells, Western blotting analysis revealed that HSF1 inhibition via small molecular including CCT361814/NXP800, an HSF1 inhibitor in phase I clinical trials, increased autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3) lipidation and decreased expression of phosphorylation of HSF1 at Ser326 and total HSF1 protein, conversely using HSF1 overexpression model reversed this effect. Inhibition of autophagy via 3-methyladenine reversed HSF1 inhibition-induced autophagy. Results from transmission electron microscopy and GFP-LC3 reporter revealed that HSF1 inhibition induced autophagosome formation and accumulation. Furthermore, HSF1 inhibition induced phosphorylation of JNK1/2 at Thr183/Tyr185 and decreased mTORC1 activity, while knockdown JNK1/2 diminished HSF1 inhibition-induced LC3 lipidation. The combination of chemotherapy drugs with HSF1 inhibitors decreased PDAC cell viability. Additionally, blocking autophagy increased HSF1 inhibition-induced apoptosis. Conclusions: In conclusion, JNK1/2 is involved in HSF1 inhibition-induced PDAC autophagy, targeting autophagy could unveil a novel treatment strategy for pancreatic cancer through targeted HSF1 inhibition. Citation Format: Shruti Ghai, Alex Young, Kuo-Hui Su. HSF1 inhibition induces pancreatic ductal adenocarcinoma autophagy through JNK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4319.

Abstract 3266: Plk1 promotes Pdcd4 degradation to enhance enzalutamide resistance in prostate cancer

Abstract The androgen receptor (AR) signaling pathway plays critical role in the development of prostate cancer (PCa), including castration-resistant prostate cancer (CRPC). As a result, androgen signaling inhibitors (ASIs) like abiraterone and enzalutamide (ENZ) have emerged as the first-line treatment for CRPC. Nevertheless, the limited success of ASIs underscores the urgency of developing new approaches for managing CRPC patients who have become unresponsive to ASIs. Programmed cell death 4 (Pdcd4) is a tumor suppressor, which is frequently down-regulated during PCa progression. It has been shown that reduced Pdcd4 expression promotes both AR-dependent and AR-independent growth in PCa. In this study, we found that down-regulation of Pdcd4 led to acquisition of ENZ resistance in PCa. We also observed that down-regulation of Pdcd4 resulted in activation of mTORC2 activity. Additionally, knockdown of Rictor, a component of mTORC2 complex, enhanced sensitivity to ENZ treatment in PCa. These findings suggested that activation of mTORC2 due to Pdcd4 loss contributes to ASI resistance in PCa. To understand how Pdcd4 is down-regulated in the PCa, we identified that polo-like kinase (Plk1) phosphorylated Pdcd4 at Ser239. Phosphorylation of Pdcd4 by Plk1 led to ubiquitin-mediated proteasomal degradation of Pdcd4. Over-expression of the phosphomimic Pdcd4(S239D) in 22Rv1 cells promoted tumor cell growth in cultured cells and mice. Taken together, our data suggest that targeting Plk1/Pdcd4/mTORC2 signaling axis holds promise as a strategy to overcome ENZ resistance in PCa. Citation Format: Qing Wang, Qiongsi Zhang, Yanquan Zhang, Xiaoqi Liu, Hsin-Sheng Yang. Plk1 promotes Pdcd4 degradation to enhance enzalutamide resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3266.

Abstract 630: The role of AKT pathway in β-catenin mutated hepatocellular carcinoma

Abstract The Wnt/β-catenin and PI3K/AKT pathways are pivotal contributors to the development of the malignant phenotype in cancer. The activated Wnt/β-catenin pathway synergizes with various signaling cascades to propel HCC formation, operating through its downstream effectors. It has been well-established that the mammalian target of rapamycin complex 2 (mTORC2) serves as an upstream regulator of AKT, orchestrating the phosphorylation and activation of AKT. Once activated, AKT phosphorylates and inhibits canonical downstream targets like forkhead box O protein (FOXO) and tuberous sclerosis complex 2 (TSC2). Studies indicated that AKT-mediated phosphorylation of TSC2 activates mTORC1, subsequently suppressing autophagy. However, there are limited data exist on the interplay between Wnt/β-catenin and AKT pathways in HCC. In this study, we sought to explore the role of the AKT pathway in β-catenin activated HCC tumors. Sleeping beauty-mediated hydrodynamic transfection was used to overexpress constitutively active forms of β-catenin (N90-β-catenin) and c-MET in the livers of mice to establish the HCC model (c-Met/β-catenin model). Notably, the knockout of Rictor, a component of the mTORC2 complex, significantly impeded tumor growth, underscoring the pivotal role of AKT in c-Met/β-catenin tumor development. AKT1 and AKT2 are the two main isoforms expressed in the liver. We determined that both AKT1 and AKT2 isoforms are critical for c-Met/β-catenin tumor growth. Intriguingly, simultaneous knockout of Rictor and activation of mTORC1 in the c-Met/β-catenin model resulted in delayed tumor growth, indicating that mTORC1 is not the sole downstream determinant of AKT signaling during c-Met/β-Catenin tumor development. Furthermore, overexpression of the activated FOXO1 (FOXO1AAA) significantly delayed the tumor growth, which highlighted the importance of FOXO1. Given the limited understanding of FOXO1 in HCC, we extended our investigation on FOXO1 to human HCC cell lines. Overexpression of FOXO1AAA notably delayed tumor growth in vitro. RNAseq analysis of FOXO1AAA-overexpressed human HCC cell lines revealed a significant upregulation of "metabolic pathways", suggesting metabolic reprogramming post FOXO1 activation. This study, for the first time, unveils the role of the AKT pathway in β-catenin-mutated HCC growth and highlights the potential therapeutic value of targeting the AKT pathway in patients with β-catenin-mutated HCC. Citation Format: Xue Wang, Xin Chen. The role of AKT pathway in β-catenin mutated hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 630.

Abstract 198: Selective targeting of endothelial mTORC1 reduces transendothelial fatty acid delivery to improve anti-tumor immunity and suppress metastatic outgrowth

Abstract Tumor metastasis requires sufficient nutrients to support outgrowth of disseminated tumor cells. Cancer cells utilize fatty acids as a key source of energy and biomass, but these nutrients impair the cytotoxic activities of T cells, serving to support metastatic progression. Blood is a rich source of fatty acids, but the vascular endothelium serves as a barrier to limit access to the surrounding tumor tissue. However, it is unclear how long-chain fatty acid delivery to early metastatic tumors is regulated. Here, we report that endothelial mTORC1 promotes transport of fatty acids into developing metastatic tumors, leading to reduced anti-tumor immune responses and improved outgrowth. Using a murine model of metastatic outgrowth in the lung, tumor burden was significantly reduced upon targeted deletion of Raptor, an essential component of the mTORC1 complex, in endothelial cells (RptorECKO). We employed a multifaceted approach using metabolomics, transcriptomics, and molecular assays to identify a Raptor/mTORC1-mediated mechanism of transendothelial fatty acid transport via RAB and CLSTN1 cargo trafficking. In vivo uptake of a fluorescent palmitic acid analog in tumor cells and T cells was reduced in RptorECKO lung metastatic tumors, which correlated with improved markers of cytotoxicity. Combination of low-dose RAD001/everolimus, which selectively inhibits mTORC1 in endothelial cells, with anti-PD1 reduces metastatic disease and impairs long-chain fatty acid uptake in T cells, corresponding to improved anti-tumor immunity. These findings describe a novel mechanism of transendothelial fatty acid transportation during metastatic outgrowth, supporting future development of therapeutic strategies to target endothelial mTORC1 activity. Citation Format: Deanna N. Edwards, Shan Wang, Wenqiang Song, Laura C. Kim, Yoonha Hwang, Jin Chen. Selective targeting of endothelial mTORC1 reduces transendothelial fatty acid delivery to improve anti-tumor immunity and suppress metastatic outgrowth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 198.

Abstract 4451: Regulation of lipid metabolism and ferroptosis by DAXX

Abstract Intracellular lipid production in cancer cells supplies lipids to synthesize cell membranes and signaling molecules during rapid cell proliferation and tumor growth. Cancer cells also utilize fatty acid oxidation (FAO) to generate ATP to meet their energy demand. Notably, lipid metabolites can inhibit and trigger ferroptosis due to iron-dependent oxidation of polyunsaturated fatty acids (PUFAs). Therefore, identifying regulators that maintain the intricate balance of lipid biosynthesis required for cell proliferation and survival is critical in cancer biology and therapy. Lipid metabolism is regulated by two oncogenic signaling pathways: the RAS-RAF-MEK-MAPK and the mammalian target of rapamycin (mTOR) pathways. About 30% of all cancers harbor constitutively active mutations in KRAS, HRAS, or NRAS, resulting in hyperactive RAS-RAF-MEK-MAPK signaling to drive tumorigenesis, metastatic progression, immune evasion, and resistance to therapy. KRAS regulates lipid uptake, lipid synthesis, and FAO. mTOR is a serine/threonine kinase acting as a key intracellular signaling hub to regulate nutrient homeostasis, metabolism, protein synthesis, and autophagy. The mTORC1 complex promotes lipogenesis. The RAS and mTOR signaling pathways exhibit both positive and negative cross-regulation. The coordinated activity of both pathways is critical to sustained tumor growth. Notably, mTORC1 signaling inhibition enhances RAS-RAF-MEK-MAPK signaling to promote cancer cell survival and proliferation. Furthermore, constitutive mTORC1 signaling induces cell death when the supply of unsaturated FAs is limited. However, the molecular link for coordinating the activity of the RAS and mTOR signaling pathways remains poorly defined. Death domain-associated protein (DAXX) is essential for mouse embryonic development and has ill-defined pro-cell survival functions. Our lab has a long-standing interest in deciphering DAXX’s complex biological functions. Our recent study shows that DAXX drives tumorigenesis by promoting lipogenic gene expression and lipid synthesis through interacting with sterol regulatory element-binding proteins SREBP1 and SREBP2 (SREBP1/2) (Mahmud et al., 2023, PMID 37045819). Unexpectedly, our new data reveal that DAXX inhibits ferroptosis. Significantly, phosphorylation via RAS signaling appears to regulate DAXX’s activity in lipid synthesis and resistance to ferroptosis. Remarkably, disruption of DAXX-mediated lipid synthesis potentiates ferroptosis due to mTORC1 activity. Based on these observations, we propose that stimulated by the RAS-RAF-MEK-MAPK oncogenic signaling, DAXX promotes tumorigenesis through upregulating genes for lipid synthesis and resistance to ferroptosis, and that the lack of DAXX-mediated lipid synthesis leads to sensitization of cells to ferroptosis due to mTORC1’s proliferative effects. (Supported by FDOH grant 23K03). Citation Format: Wenlin Yang, Nikee Awasthee, Qi Chen, Seth Hale, Daiqing Liao. Regulation of lipid metabolism and ferroptosis by DAXX [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4451.

MTOR hyperactivity and RICTOR amplification as targets for personalized treatments in malignancies.

The increasing knowledge of molecular alterations in malignancies, including mutations and regulatory failures in the mTOR (mechanistic target of rapamycin) signaling pathway, highlights the importance of mTOR hyperactivity as a validated target in common and rare malignancies. This review summarises recent findings on the characterization and prognostic role of mTOR kinase complexes (mTORC1 and mTORC2) activity regarding differences in their function, structure, regulatory mechanisms, and inhibitor sensitivity. We have recently identified new tumor types with RICTOR (rapamycin-insensitive companion of mTOR) amplification and associated mTORC2 hyperactivity as useful potential targets for developing targeted therapies in lung cancer and other newly described malignancies. The activity of mTOR complexes is recommended to be assessed and considered in cancers before mTOR inhibitor therapy, as current first-generation mTOR inhibitors (rapamycin and analogs) can be ineffective in the presence of mTORC2 hyperactivity. We have introduced and proposed a marker panel to determine tissue characteristics of mTOR activity in biopsy specimens, patient materials, and cell lines. Ongoing phase trials of new inhibitors and combination therapies are promising in advanced-stage patients selected by genetic alterations, molecular markers, and/or protein expression changes in the mTOR signaling pathway. Hopefully, the summarized results, our findings, and the suggested characterization of mTOR activity will support therapeutic decisions.