Xiuyun Jiang

Tuberin Regulates E-Cadherin Localization: Implications in Epithelial-Mesenchymal Transition

The tuberous sclerosis complex 2 (TSC2) gene encodes the protein tuberin, which functions as a key negative regulator of both mammalian target of rapamycin (mTOR) C1-dependent cell growth and proliferation. Loss-of-function mutations of TSC2 result in mTORC1 hyperactivity and predispose individuals to both tuberous sclerosis and lymphangioleiomyomatosis. These overlapping diseases have in common the abnormal proliferation of smooth muscle-like cells. Although the origin of these cells is unknown, accumulating evidence suggests that a metastatic mechanism may be involved, but the means by which the mTOR pathway contributes to this disease process remain poorly understood. In this study, we show that tuberin regulates the localization of E-cadherin via an Akt/mTORC1/CLIP170-dependent, rapamycin-sensitive pathway. Consequently, Tsc2(-/-) epithelial cells display a loss of plasma membrane E-cadherin that leads to reduced cell-cell adhesion. Under confluent conditions, these cells detach, grow in suspension, and undergo epithelial-mesenchymal transition (EMT) that is marked by reduced expression levels of both E-cadherin and occludin and increased expression levels of both Snail and smooth muscle actin. Functionally, the Tsc2(-/-) cells demonstrate anchorage-independent growth, cell scattering, and anoikis resistance. Human renal angiomyolipomas and lymphangioleiomyomatosis also express markers of EMT and exhibit an invasive phenotype that can be interpreted as consistent with EMT. Together, these results suggest a novel relationship between TSC2/mTORC1 and the E-cadherin pathways and implicate EMT in the pathogenesis of tuberous sclerosis complex-related diseases.

The Tuberous Sclerosis Complex Regulates Trafficking of Glucose Transporters and Glucose Uptake

Human cancers often display an avidity for glucose, a feature that is exploited in clinical staging and response monitoring by using (18)F-fluoro-deoxyglucose (FDG) positron emission tomography. Determinants of FDG accumulation include tumor blood flow, glucose transport, and glycolytic rate, but the underlying molecular mechanisms are incompletely understood. The phosphoinositide-3 kinase/Akt/mammalian target of rapamycin complex (mTORC) 1 pathway has been implicated in this process via the hypoxia-inducible factor alpha-dependent expression of vascular endothelial growth factor and glycolytic enzymes. Thus, we predicted that tumors with elevated mTORC1 activity would be accompanied by high FDG uptake. We tested this hypothesis in eight renal angiomyolipomas in which the loss of tuberous sclerosis complex (TSC) 1/2 function gave rise to constitutive mTORC1 activation. Surprisingly, these tumors displayed low FDG uptake on positron emission tomography. Exploring the underlying mechanisms in vitro revealed that Tsc2 regulates the membrane localization of the glucose transporter proteins (Glut)1, Glut2, and Glut4, and, therefore, glucose uptake. Down-regulation of cytoplasmic linker protein 170, an mTOR effector, rescued Glut4 trafficking in Tsc2(-/-) cells, whereas up-regulation of Akt activity in these cells was insufficient to redistribute Glut4 to the plasma membrane. The effect of mTORC1 on glucose uptake was confirmed using a liver-specific Tsc1- deletion mouse model in which FDG uptake was reduced in the livers of mutant mice compared with wild-type controls. Together, these data show that mTORC1 activity is insufficient for increased glycolysis in tumors and that constitutive mTOR activity negatively regulates glucose transporter trafficking.

Regulation of microtubule-dependent protein transport by the TSC2/mammalian target of rapamycin pathway

Protein transport plays a critical role in the interaction of the cell with its environment. Recent studies have identified TSC1 and TSC2, two tumor suppressor genes involved in tuberous sclerosis complex, as regulators of the mammalian target of rapamycin (mTOR) pathway. Cells deficient in TSC1 or TSC2 possess high levels of Rheb-GTP resulting in constitutive mTOR activation. We have shown previously that the TSC1/TSC2 complex is involved in post-Golgi transport of VSVG and caveolin-1 in mammalian cells. Here, we show that modulation of mTOR activity affects caveolin-1 localization and that this effect is independent of p70S6K. Tsc1- and Tsc2-null cells exhibit abnormal caveolin-1 localization that is accompanied by disorganized microtubules in the subcortical region. Analyses of green fluorescent protein-EB1 and tubulin in live mutant cells suggest a failure of the plus-ends to sense cortical signals and to halt microtubule growth. Down-regulation of CLIP-170, a putative mTOR substrate with microtubule-binding properties, rescued the abnormal microtubule arrangement and caveolin-1 localization in Tsc2-/- cells. Together, these findings highlight a novel role of the TSC2/mTOR pathway in regulating microtubule-dependent protein transport.

Biopolymers codelivering engineered T cells and STING agonists can eliminate heterogeneous tumors

Therapies using T cells that are programmed to express chimeric antigen receptors (CAR T cells) consistently produce positive results in patients with hematologic malignancies. However, CAR T cell treatments are less effective in solid tumors for several reasons. First, lymphocytes do not efficiently target CAR T cells; second, solid tumors create an immunosuppressive microenvironment that inactivates T cell responses; and third, solid cancers are typified by phenotypic diversity and thus include cells that do not express proteins targeted by the engineered receptors, enabling the formation of escape variants that elude CAR T cell targeting. Here, we have tested implantable biopolymer devices that deliver CAR T cells directly to the surfaces of solid tumors, thereby exposing them to high concentrations of immune cells for a substantial time period. In immunocompetent orthotopic mouse models of pancreatic cancer and melanoma, we found that CAR T cells can migrate from biopolymer scaffolds and eradicate tumors more effectively than does systemic delivery of the same cells. We have also demonstrated that codelivery of stimulator of IFN genes (STING) agonists stimulates immune responses to eliminate tumor cells that are not recognized by the adoptively transferred lymphocytes. Thus, these devices may improve the effectiveness of CAR T cell therapy in solid tumors and help protect against the emergence of escape variants.

Akt and mTORC1 Have Different Roles During Liver Tumorigenesis in Mice

BACKGROUND & AIMS Phosphatidylinositide 3-kinase (PI3K) is deregulated in many human tumor types, including primary liver malignancies. The kinase v-akt murine thymoma viral oncogene homolog 1 (Akt) and mammalian target of rapamycin complex (mTORC1) are effectors of PI3K that promote cell growth and survival, but their individual roles in tumorigenesis are not well defined. METHODS In livers of albumin (Alb)-Cre mice, we selectively deleted tuberous sclerosis (Tsc)1, a negative regulator of Ras homolog enriched in brain and mTORC1, along with Phosphatase and tensin homolog (Pten), a negative regulator of PI3K. Tumor tissues were characterized by histologic and biochemical analyses. RESULTS The Tsc1fl/fl;AlbCre, Ptenfl/fl;AlbCre, and Tsc1fl/fl;Ptenfl/fl;AlbCre mice developed liver tumors that differed in size, number, and histologic features. Livers of Tsc1fl/fl;AlbCre mice did not develop steatosis; tumors arose later than in the other strains of mice and were predominantly hepatocellular carcinomas. Livers of the Ptenfl/fl;AlbCre mice developed steatosis and most of the tumors that formed were intrahepatic cholangiocarcinomas. Livers of Tsc1fl/fl;Ptenfl/fl;AlbCre formed large numbers of tumors, of mixed histologies, with the earliest onset of any strain, indicating that loss of Tsc1 and Pten have synergistic effects on tumorigenesis. In these mice, the combination of rapamycin and MK2206 was more effective in reducing liver cell proliferation and inducing cell death than either reagent alone. Tumor differentiation correlated with Akt and mTORC1 activities; the ratio of Akt:mTORC1 activity was high throughout the course of intrahepatic cholangiocarcinomas development and low during hepatocellular carcinoma development. Compared with surrounding nontumor liver tissue, tumors from all 3 strains had increased activities of Akt, mTORC1, and mitogen-activated protein kinase and overexpressed fibroblast growth factor receptor 1. Inhibition of fibroblast growth factor receptor 1 in Tsc1-null mice suppressed Akt and mitogen-activated protein kinase activities in tumor cells. CONCLUSIONS Based on analyses of knockout mice, mTORC1 and Akt have different yet synergistic effects during the development of liver tumors in mice.

Long-lived pancreatic ductal adenocarcinoma slice cultures enable precise study of the immune microenvironment

ABSTRACT Pancreatic ductal adenocarcinoma (PDA) remains a deadly disease that is rarely cured, despite many recent successes with immunotherapy for other malignancies. As the human disease is heavily infiltrated by effector T cells, we postulated that accurately modeling the PDA immune microenvironment would allow us to study mechanisms of immunosuppression that could be overcome for therapeutic benefit. Using viable precision-cut slices from fresh PDA, we developed an organotypic culture system for this purpose. We confirmed that cultured slices maintain their baseline morphology, surface area, and microenvironment after at least 6 d in culture, and demonstrated slice survival by MTT assay and by immunohistochemistry staining with Ki-67 and cleaved-Caspase-3 antibodies. Immune cells, including T cells (CD3+, CD8+, and FOXP3+) and macrophages (CD68+, CD163+ and HLA-DR+), as well as stromal myofibroblasts (αSMA+) were present throughout the culture period. Global profiling of the PDA proteome before and after 6 d slice culture indicated that the majority of the immunological proteins identified remain stable during the culture process. Cytotoxic effects of drug treatment (staurosporine, STS and cycloheximide, CHX) on PDA slices culture confirmed that this system can be used to assess functional response and cell survival following drug treatment in both a treatment time- and dose-dependent manner. Using multicolor immunofluorescence, we stained live slices for both cancer cells (EpCAM+) and immune cells (CD11b+ and CD8+). Finally, we confirmed that autologous CFSE-labeled splenocytes readily migrate into co-cultured tumor slices. Thus, our present study demonstrates the potential to use tumor slice cultures to study the immune microenvironment of PDA.