Ras Homolog Enriched In Brain Research Articles

Genetic Regulation of Fate Decisions in Therapeutic T Cells to Enhance Tumor Protection and Memory Formation.

A key challenge in the field of T-cell immunotherapy for cancer is creating a suitable platform for promoting differentiation of effector cells while at the same time enabling self-renewal needed for long-term memory. Although transfer of less differentiated memory T cells increases efficacy through greater expansion and persistence in vivo, the capacity of such cells to sustain effector functions within immunosuppressive tumor microenvironments may still be limiting. We have therefore directly compared the impact of effector versus memory differentiation of therapeutic T cells in tumor-bearing mice by introducing molecular switches that regulate cell fate decisions via mTOR. Ectopic expression of RAS homolog enriched in brain (RHEB) increased mTORC1 signaling, promoted a switch to aerobic glycolysis, and increased expansion of effector T cells. By rapidly infiltrating tumors, RHEB-transduced T cells significantly reduced the emergence of immunoedited escape variants. In contrast, expression of proline-rich Akt substrate of 40 kDa (PRAS40) inhibited mTORC1, promoted quiescence, and blocked tumor infiltration. Fate mapping studies following transient expression of PRAS40 demonstrated that mTORC1(low) T cells made no contribution to initial tumor control but instead survived to become memory cells proficient in generating recall immunity. Our data support the design of translational strategies for generating heterogeneous T-cell immunity against cancer, with the appropriate balance between promoting effector differentiation and self-renewal. Unlike pharmacologic inhibitors, the genetic approach described here allows for upregulation as well as inhibition of the mTORC1 pathway and is highly selective for the therapeutic T cells without affecting systemic mTORC1 functions.

MTORC1 and mTORC2 selectively regulate CD8⁺ T cell differentiation.

Activation of mTOR-dependent pathways regulates the specification and differentiation of CD4+ T effector cell subsets. Herein, we show that mTOR complex 1 (mTORC1) and mTORC2 have distinct roles in the generation of CD8+ T cell effector and memory populations. Evaluation of mice with a T cell-specific deletion of the gene encoding the negative regulator of mTORC1, tuberous sclerosis complex 2 (TSC2), resulted in the generation of highly glycolytic and potent effector CD8+ T cells; however, due to constitutive mTORC1 activation, these cells retained a terminally differentiated effector phenotype and were incapable of transitioning into a memory state. In contrast, CD8+ T cells deficient in mTORC1 activity due to loss of RAS homolog enriched in brain (RHEB) failed to differentiate into effector cells but retained memory characteristics, such as surface marker expression, a lower metabolic rate, and increased longevity. However, these RHEB-deficient memory-like T cells failed to generate recall responses as the result of metabolic defects. While mTORC1 influenced CD8+ T cell effector responses, mTORC2 activity regulated CD8+ T cell memory. mTORC2 inhibition resulted in metabolic reprogramming, which enhanced the generation of CD8+ memory cells. Overall, these results define specific roles for mTORC1 and mTORC2 that link metabolism and CD8+ T cell effector and memory generation and suggest that these functions have the potential to be targeted for enhancing vaccine efficacy and antitumor immunity.

PDK4 Protein Promotes Tumorigenesis through Activation of cAMP-response Element-binding Protein (CREB)-Ras Homolog Enriched in Brain (RHEB)-mTORC1 Signaling Cascade

Mechanistic target of rapamycin (mTOR) integrates multiple extracellular and intracellular signals to regulate cell growth and survival. Hyperactivation of mTOR has been observed in various cancers. Regulation of mTOR activity is thus of importance in physiological processes and tumor development. Here, we present pyruvate dehydrogenase kinase 4 (PDK4) as a novel regulator of mTORC1 signaling. mTORC1 activity was augmented with PDK4 overexpression and reduced by PDK4 suppression in various cell lines. Furthermore, PDK4 bound to cAMP-response element-binding protein (CREB) and prevented its degradation. The enhanced CREB consequently transactivated the expression of Ras homolog enriched in brain (RHEB), a direct key activator of mTORC1, independent of AMP-activated protein kinase or tuberous sclerosis complex protein 2. PDK4 potentiated the mTORC1 effectors hypoxia-inducible factor 1α and pyruvate kinase isozymes M2 and promoted aerobic glycolysis (Warburg effect). Knockdown of PDK4 suppressed the tumor development of cancer cells with activated mTORC1. The abundance of PDK4 dictated the responsiveness of cells to the mTOR inhibitor, rapamycin. Combinatory suppression of mTOR and PDK4 exerted synergistic inhibition on cancer cell proliferation. Therefore, PDK4 promotes tumorigenesis through activation of the CREB-RHEB-mTORC1 signaling cascade.

Novel mutations and role of the LKB1 gene as a tumor suppressor in renal cell carcinoma.

The tumor suppressor LKB1 gene is a master kinase and inhibits mammalian target of rapamycin (mTOR) by activating AMP-activated protein kinase (AMPK) and AMPK-related kinases. LKB1 is a critical intermediate in the mTOR signaling pathway, and mutations of the LKB1 gene have been implicated in the development of different tumor types. Recent evidence indicates that LKB1 alterations contribute to cancer progression and metastasis by modulating vascular endothelial growth factor (VEGF) production. The Ras homolog enriched in brain (RHEB) protein is a component of the mTOR pathway and functions as a positive regulator of mTOR. However, the mechanisms and effectors of RHEB in mTOR signaling are not well known. In this study, we analyzed the expression of RHEB and HIF1α genes in correlation with LKB1 gene mutations. All coding exons and exon/intron boundaries of the LKB1 gene were analyzed by direct sequencing in 77 renal cell carcinoma (RCC) tumors and 62 matched noncancerous tissue samples. In 51.6% of the patients, ten different mutations including four novel mutations in the coding sequences and six single nucleotide substitutions in the introns were observed. Rheb and HIF1α expression levels were not statistically different between the tumor and corresponding noncancerous tissue samples. However, expression of the Rheb gene was upregulated in the tumor samples carrying the intron 2 (+24 G→T) alteration. Association between the gene expression and tissue protein levels was also analyzed for HIF1α in a subgroup of patients, and a high correlation was confirmed. Our results indicate that the LKB1 gene is frequently altered in RCC and may play a role in RCC progression.

Tuning Of mTOR In Therapeutic T Cells As A Strategy To Manufacture Effector and Memory T Cells

Retroviral gene transfer of T cell receptors (TCRs) or chimeric-antigen receptors has been successfully used to redirect T cell specificity to tumor antigens and has shown promising results in clinical trials for patients with melanoma and B cell malignancies. Successful therapy relies not only on the generation of an efficient effector response but also in the ability of T cells to engraft and persist during and after cancer rejection. Yet, both aspects can be compromised. On the one hand, effector T cells can be disarmed in vivo by the immunosuppressive environment of the tumor, where the depletion of key nutrients (glucose, arginine, etc.) and the predominance of inhibitory signals (TGF-β, PD-L1, etc.) dampen their functions. On the other hand, current retroviral gene transfer protocols require T cells to be activated and expanded ex vivo,driving terminal effector differentiation at the expense of the capacity for self-renewal (“stemness”). This may prevent the generation of long-term protective memory and compromise therapeutic success. The mechanistic target of rapamycin (mTOR) pathway has recently emerged as a driver of effector differentiation in CD8 T cells by acting as a signalling bridge between extracellular stimuli and effector differentiation. Whilst high doses of rapamycin are known to be immunosuppressive, a low dose regimen increases memory output after a viral challenge without impairing viral clearance. These and other studies suggest that mTOR can be used as a rheostat to regulate T cell differentiation to clinical advantage. Using a mouse model of TCR gene therapy we aimed to produce T cells in which the mTOR pathway is either hyperactive, in order to generate ‘super-effectors’ that can function in the tumor microenvironment, or hypoactive in order to preserve “stemness”, increase engraftment and produce better recall immunity. To this purpose we transferred genes into therapeutic T cells encoding either (1) an mTOR activator, RHEB (Ras homolog enriched in brain) or (2) an mTOR inhibitor, PRAS40 (proline-rich Akt substrate 40 kDa). Upon stimulation in vitro,the phosphorylation of ribosomal protein S6, an mTOR downstream target, was increased in RHEB-expressing cells and decreased in PRAS40-expressing cells. RHEB T cells demonstrated a greater propensity than controls for blast formation, lower expression of L-selectin, increased IFN-g production and resistance to TGF-b. In contrast, PRAS40 T cells were smaller in size, expressed higher surface levels of L-selectin and had reduced but not ablated production of IFN-g, IL-2 and TNF-a production. In vivo, RHEB T cells co-transduced with a tumor-specific TCR showed increased initial expansion that was associated with increased tumor protection. However, this was followed by a steep decrease in T cell numbers compared to control cells, with preliminary experiments indicating that RHEB cells produced a less robust re-call response upon antigenic re-challenge. In contrast, PRAS40 cells failed to expand in vivo or to infiltrate tumors, resulting in complete lack of protection. Yet, the few PRAS40 cells remaining in circulation were predominately high for L-selectin, IL-7Ra and Sca-1 (markers associated with ‘stemness’). These results demonstrated that constitutive suppression of mTOR was of no therapeutic advantage. To circumvent this, we developed a doxycycline (DOX)-inducible PRAS40 vector in which mTOR suppression in T cells can be temporally controlled in vivo. Unlike the constitutive vector used previously, with the DOX-ON system the level of mTOR suppression was lower and furthermore, could be applied only during the period of antigen exposure. Thus, when DOX was added during tumor challenge, PRAS40-expressing T cells could reject tumor as well as controls. However, after DOX was withdrawn and transgene expression turned off, increased numbers of therapeutic T cells were found in peripheral blood. When re-challenged, these T cells produced a stronger recall response relative to cells in which mTOR had not been suppressed initially. Thus, our results show that genetic manipulation of mTOR can be used to alter the intrinsic properties of T cells and direct either their effector or memory differentiation. This approach maybe useful in designing better therapeutic immunotherapeutic strategies according to the type of T cell response required in vivo. Disclosures: No relevant conflicts of interest to declare.

Expressional Difference of RHEB, HDAC1, and WEE1 Proteins in the Stromal Tumors of the Breast and Their Significance in Tumorigenesis

BackgroundFibroadenoma (FA) and phyllodes tumor (PT) are stromal tumors of breast and are histologically similar. There are no established differences in tumorigenesis and oncogene expression among them. Ras homolog enriched in brain (RHEB) plays an important role in cell growth and cell-cycle control, histone deacetylase 1 (HDAC1) is an important factor in breast tumor progression and prognosis, and WEE1 homolog (WEE1) functions as a tumor suppressor. No studies on the expressional differences of these proteins in FA and PT have been reported to date.MethodsThe expression of these proteins in FA, PT, and normal breast was compared. We used 102 cases of FA and 25 cases of benign PT.ResultsIn epithelial cells, the expression of RHEB, HDAC1, and WEE1 was lowest in PT, higher in FA, and most enhanced in normal breast. In addition, the expression of RHEB and HDAC1 was higher in the stromal cells of PT than in FA and normal breast.ConclusionsBoth epithelial and stromal cells of FA and PT express these proteins, which indicates that epithelial cells play an important role in the development of stromal tumors. In addition, the expressional differences of these proteins may be associated with the tumorigenesis of breast stromal tumors.

Targeting phosphatidylinositol 3 kinase (PI3K)-Akt beyond rapalogs

The activation of the phosphatidylinositol 3 kinase (PI3K)-Akt pathway is a known causal mechanism of oncogenesis and resistance to cancer treatments. The process of PI3K-Akt pathway activation is complex and includes receptor tyrosine kinase(RTK) activation, PIK3CA mutations, loss of phosphatase and tensin homolog (PTEN), Akt mutations, tuberous sclerosis complex (TSC) mutations, and Ras homologue enriched in brain (RHEB) gene amplifications. The blockage of mammalian target of rapamycin (mTOR), the key downstream pathway protein, has been successful in selected cancer types, with mTOR-targeting agents available for clinical use. Other novel drugs blocking this pathway such as PI3K inhibitors, Akt inhibitors and PDK-1 inhibitors are currently only available for investigational use, but have shown promise as cancer therapies in both preclinical and early phase clinical studies. The newer generations of these inhibitors are more specific and have improved potency and safety. The combinations of targeted treatments against this pathway, blocking multiple different steps, are under preliminary investigation. Further research is needed to identify the biomarkers that predict treatment response and resistance in order to optimize personalized medicine.

The Farnesyl Transferase Inhibitor Lonafarnib Inhibits mTOR Signaling and Enforces Sorafenib-Induced Apoptosis in Melanoma Cells

Farnesyl transferase inhibitors (FTIs) inhibit the farnesylation of proteins, including RAS and RHEB (Ras homolog enriched in brain). RAS signals to the RAF-MEK-ERK (MAPK) and PI3K-AKT-mTOR (AKT) signaling pathways, which have a major role in melanoma progression. RHEB positively regulates mammalian target of rapamycin (mTOR). We investigated the effects of the FTI lonafarnib alone and in combination with MAPK (mitogen-activated protein kinase) or AKT (acutely transforming retrovirus AKT8 in rodent T-cell lymphoma) pathway inhibitors on proliferation, survival, and invasive tumor growth of melanoma cells. Lonafarnib alone did not sufficiently inhibit melanoma cell growth. Combinations of lonafarnib with AKT pathway inhibitors did not significantly increase melanoma cell growth inhibition. In contrast, combinations of lonafarnib with MAPK pathway inhibitors yielded additional growth-inhibiting effects. In particular, the combination of the FTI lonafarnib with the pan-RAF inhibitor sorafenib synergistically inhibited melanoma cell growth, significantly enhanced sorafenib-induced apoptosis, and completely suppressed invasive tumor growth in monolayer and organotypic cultures, respectively. Apoptosis induction was associated with upregulation of the endoplasmic reticulum stress-related transcription factors p8 and CHOP (CAAT/enhancer binding protein (C/EBP) homologous protein), and downregulation of the antiapoptotic Bcl-2 (B-cell lymphoma-2) family protein Mcl-1(myeloid cell leukemia 1). Lonafarnib did not affect MAPK and AKT but did affect mTOR signaling. Together, these findings suggest that the FTI lonafarnib inhibits mTOR signaling and enforces sorafenib-induced apoptosis in melanoma cells and may therefore represent an effective alternative for melanoma treatment.

Tumor hypoxia and genetic alterations in sporadic cancers

The cancer genome contains many gene alterations. How cancer cells acquire these alterations is a matter for discussion. One hypothesis is that cancer cells obtain mutations in genome stability genes at an early stage of tumor development, which results in genetic instability and generates a gene pool that enhances cellular proliferation and survival. Another hypothesis puts its emphasis on the natural selection of gene mutations for fitness. Recent data for systematic cancer genome sequencing shows that mutations in stability genes are rare in human sporadic cancers. Instead, many “passenger” mutations that do not drive the carcinogenesis process have been found in the cancer genome. Both the hypotheses mentioned above fall short in explaining recent data. Recently, many studies demonstrate the role of the tumor microenvironment, especially hypoxia and reoxygenation, in genetic instability. In this review, literature will be presented which supports a third hypothesis, i.e. that hypoxia/re-oxygenation induces genetic instability.

Mammalian target of rapamycin activator RHEB is frequently overexpressed in human carcinomas and is critical and sufficient for skin epithelial carcinogenesis.

Small GTPase Ras homologue enriched in brain (RHEB) binds and activates the key metabolic regulator mTORC1, which has an important role in cancer cells, but the role of RHEB in cancer pathogenesis has not been shown. By performing a meta-analysis of published cancer cytogenetic and transcriptome databases, we defined a gain of chromosome 7q36.1-q36.3 containing the RHEB locus, an overexpression of RHEB mRNA in several different carcinoma histotypes, and an association between RHEB upregulation and poor prognosis in breast and head and neck cancers. To model gain of function in epithelial malignancy, we targeted Rheb expression to murine basal keratinocytes of transgenic mice at levels similar to those that occur in human squamous cancer cell lines. Juvenile transgenic epidermis displayed constitutive mTORC1 pathway activation, elevated cyclin D1 protein, and diffuse skin hyperplasia. Skin tumors subsequently developed with concomitant stromal angio-inflammatory foci, evidencing induction of an epidermal hypoxia-inducible factor-1 transcriptional program, and paracrine feed-forward activation of the interleukin-6-signal transducer and activator of transcription 3 pathway. Rheb-induced tumor persistence and neoplastic molecular alterations were mTORC1 dependent. Rheb markedly sensitized transgenic epidermis to squamous carcinoma induction following a single dose of Ras-activating carcinogen 7,12-dimethylbenz(a)anthracene. Our findings offer direct evidence that RHEB facilitates multistage carcinogenesis through induction of multiple oncogenic mechanisms, perhaps contributing to the poor prognosis of patients with cancers overexpressing RHEB.