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The multidomain protein encoded by the Tumor Susceptibility Gene 101 ( TSG101 ) is ubiquitously expressed and is suggested to function in diverse intracellular processes. In this review, we provide a succinct overview of the main structural features of the protein and their suggested roles in molecular and cellular functions. We then summarize, in more detail, key findings from studies using genetically engineered animal models that demonstrate essential functions of TSG101 in cell proliferation and survival, normal tissue homeostasis, and tumorigenesis. Despite studies on cell lines that provide insight into the molecular underpinnings by which TSG101 might function as a negative growth regulator, a biologically significant role of TSG101 as a tumor suppressor has yet to be confirmed using genuine in vivo cancer models. More recent observations from several cancer research teams suggest that TSG101 might function as an oncoprotein. A potential role of post-translational mechanisms that control the expression of the TSG101 protein in cancer is being discussed. In the final section of the review, we summarize critical issues that need to be addressed to gain a better understanding of biologically significant roles of TSG101 in cancer., Competing Interests: The authors declare no conflict of interest.

Neuropilin-2 (NRP2) is a non-tyrosine kinase receptor frequently overexpressed in various malignancies, where it has been implicated in promoting many protumorigenic behaviors, such as imparting therapeutic resistance to metastatic cancer cells. Here, we report a novel function of NRP2 as a regulator of endocytosis, which is enhanced in cancer cells and is often associated with increased metastatic potential and drug resistance. We found that NRP2 depletion in human prostate and pancreatic cancer cells resulted in the accumulation of EEA1/Rab5-positive early endosomes concomitant with a decrease in Rab7-positive late endosomes, suggesting a delay in early-to-late endosome maturation. NRP2 depletion also impaired the endocytic transport of cell surface EGFR, arresting functionally active EGFR in endocytic vesicles that consequently led to aberrant ERK activation and cell death. Mechanistic investigations revealed that WD-repeat- and FYVE-domain-containing protein 1 (WDFY1) functioned downstream of NRP2 to promote endosome maturation, thereby influencing the endosomal trafficking of EGFR and the formation of autolysosomes responsible for the degradation of internalized cargo. Overall, our results indicate that the NRP2/WDFY1 axis is required for maintaining endocytic activity in cancer cells, which supports their oncogenic activities and confers drug resistance. Therefore, therapeutically targeting endocytosis may represent an attractive strategy to selectively target cancer cells in multiple malignancies., (©2015 American Association for Cancer Research.)

Understanding the molecular mechanisms promoting therapy resistance is important. Previously, we reported that VEGFC can promote cancer cell survival during stress via interaction with its receptor NRP2. While examining the molecular mechanisms involved in this survival, we performed a microarray study in which we identified two genes, WDFY1 and LAMP2, which have been suggested to function in autophagy. Our subsequent studies further confirmed the regulation of autophagy by the VEGFC-NRP2 axis in cancer during starvation- and chemotherapy-induced stress. We are currently in the process of determining the mechanism(s) through which WDFY1 and LAMP2 control autophagy; however, we did observe an increase in MTOR complex 1 (MTORC1) activity after the depletion of the VEGFC-NRP2 axis. It would therefore be interesting to study whether WDFY1 and LAMP2 can influence MTORC1 activity and regulate autophagy. Taken together, our data suggest that targeting the VEGFC-NRP2 axis in combination with chemotherapy could be an effective treatment for advanced cancers.

A major contributor to cancer mortality is recurrence and subsequent metastatic transformation following therapeutic intervention. Therefore, in order to develop new treatment modalities and improve the efficacy of current ones, it is important to understand the molecular mechanisms that promote resistance to therapy in cancer cells. One pathway contributing to therapy resistance is autophagy, a self-digestive process that can eliminate unnecessary or damaged organelles to protect cancer cells from death. We have found that the VEGF-C/NRP-2 axis is involved in the activation of autophagy, which helps cancer cell survival following treatment. Inhibition of mTOR complex 1 activity by this axis is the underlying mechanism for the activation of autophagy. Furthermore, we identified two VEGF-C/NRP-2-regulated genes, LAMP-2 and WDFY-1, that have previously been suggested to participate in autophagy and vesicular trafficking. Upregulation of WDFY-1 following VEGF-C or NRP-2 depletion contributes to cytotoxic drug-mediated cell death. Together, these data suggest a link between the VEGF-C/NRP-2 axis and cancer cell survival despite the presence of chemotherapy-induced stress. Effective targeting of this pathway may lead to the development of new cancer therapies.

The Tumor Susceptibility Gene 101 (Tsg101) encodes a multi-domain protein that mediates a variety of molecular and biological processes including the trafficking and lysosomal degradation of cell surface receptors. Conventional and conditional knockout models have demonstrated an essential requirement of this gene for cell cycle progression and cell viability, but the consequences of a complete ablation of Tsg101 on intracellular processes have not been examined to date. In this study, we employed mouse embryonic fibroblasts that carry two Tsg101 conditional knockout alleles to investigate the expression of ErbB receptor tyrosine kinases as well as stress-induced intracellular processes that are known to be associated with a defect in growth and cell survival. The conditional deletion of the Tsg101 gene in this well-controlled experimental model resulted in a significant reduction in the steady-state levels of the EGFR and ErbB2 but a stress-induced elevation in the phosphorylation of mitogen activated protein (MAP) kinases independent of growth factor stimulation. As part of an integrated stress response, Tsg101-deficient cells exhibited extensive remodeling of actin filaments and greatly enlarged lysosomes that were enriched with the autophagy-related protein LC3. The increase in the transcriptional activation and expression of LC3 and its association with Lamp1-positive lysosomes in a PI3K-dependent manner suggest that Tsg101 knockout cells utilize autophagy as a survival mechanism prior to their ultimate death. Collectively, this study shows that a knockout of the Tsg101 gene causes complex intracellular changes associated with stress response and cell death. These multifaceted alterations need to be recognized as they have an impact on defining particular functions for Tsg101 in processes such as signal transduction and lysosomal/endosomal trafficking.

Active Src localization at focal adhesions (FAs) is essential for cell migration. How this pool is linked mechanistically to the large pool of Src at late endosomes (LEs)/lysosomes (LY) is not well understood. Here, we used inducible Tsg101 gene deletion, TSG101 knockdown, and dominant-negative VPS4 expression to demonstrate that the localization of activated cellular Src and viral Src at FAs requires the endosomal-sorting complexes required for transport (ESCRT) pathway. Tsg101 deletion also led to impaired Src-dependent activation of STAT3 and focal adhesion kinase and reduced cell migration. Impairment of the ESCRT pathway or Rab7 function led to the accumulation of active Src at aberrant LE/LY compartments followed by its loss. Analyses using fluorescence recovery after photo-bleaching show that dynamic mobility of Src in endosomes is ESCRT pathway-dependent. These results reveal a critical role for an ESCRT pathway-dependent LE/LY trafficking step in Src function by promoting localization of active Src to FAs.

Previous studies reported that the Tumor Susceptibility Gene 101 (TSG101) is upregulated in selected human malignancies, and the expression of exogenous Tsg101 was suggested to transform immortalized fibroblasts in culture. To date, the potential oncogenic properties of Tsg101 have not been examined in vivo owing to the lack of appropriate model systems. In this study, we show that Tsg101 is highly expressed in a subset of invasive human breast cancers. Based on this observation, we generated the first transgenic mouse model with a targeted overexpression of Tsg101 in the developing mammary gland to test whether exogenous Tsg101 is capable of initiating tumorigenesis. Normal functionality of exogenous Tsg101 was tested by rescuing the survival of Tsg101-deficient mammary epithelial cells in conditional knockout mice. The overexpression of Tsg101 resulted in increased phosphorylation of the epidermal growth factor receptor and downstream activation of MAP kinases. Despite an increase in the activation of these signal transducers, the mammary gland of females expressing exogenous Tsg101 developed normally throughout the reproductive cycle. In aging females, the overexpression of Tsg101 seemed to increase the susceptibility of mammary epithelia toward malignant transformation. However, owing to the long latency of tumor formation and the sporadic occurrence of bona fide mammary cancers, we conclude that the Tsg101 protein has only weak oncogenic properties. Instead of cancer initiation, it is therefore likely that Tsg101 plays a more predominant role in the progression of a subset of spontaneously arising breast cancers.

Malacoplakia is a granulomatous disease that most frequently involves the urinary tract but also may involve the genital tract, gastrointestinal tract and retroperitoneum. It is believed to be infectious in origin, secondary to a deficiency of intracellular lysosomal digestion, and heretofore considered a chronic problem. We report a case of malacoplakia of the bladder, which was treated successfully with a combination of bethanechol, trimethoprim-sulfamethoxazole and ascorbic acid.