Biological and intracellular functions of Tumor susceptibility gene 101

The Tumor susceptibility gene 101 ( Tsg101) has been proposed to be expressed in several human cancers, but its expression level in breast tumors has not been thoroughly examined. Therefore, we began our investigation into the function of Tsg101 by determining its expression levels in benign and invasive breast lesions as well as in normal tissue. In these studies we found that Tsg101 was highly upregulated in a subset of invasive tumors and that this upregulation was most distinct in tumor cells that had invaded the surrounding stroma. Subsequent studies in an overexpression model of Tsg101 revealed that mammary gland tumorigenesis occurred with a higher frequency. Tsg101 is a multidomain protein that has been suggested to function in several cellular processes including cell cycle regulation and the degradation of mitogenic receptors. Tsg101 had been suggested to regulate cell cycle progression via direct interaction with the cell-cycle regulators p21Cip1 and MDM2. Tsg101 has also been suggested to regulate the degradation of mitogenic receptors such as EGFR and ErbB2. Although some of the intracellular functions of Tsg101 have been studied in human cell lines using siRNA-based knock down assays, the effects of a complete ablation of this protein on both the cell cycle machinery and the endosomal degradation of cell-surface receptors have not been examined. We previously generated Tsg101 conditional knock-out mice using the Cre/lox system to study the biological functions of the Tsg101 gene in vivo. Subsequently, we derived immortalized, conditional knockout cell lines from these mice in which Tsg101 can be deleted in culture using retroviral-based expression of Cre recombinase. Following the excision of both Tsg101 alleles, we assayed the effect of Tsg101 ablation on cell cycle progression, cell survival and the stability of cell cycle regulatory proteins such as MDM2. In these studies, we observed that the loss of Tsg101 led to a p53-dependent cell cycle arrest. Furthermore, we determined that Tsg101 does not regulate cell cycle progression via direct interaction with regulatory members of the cell cycle machinery. Therefore, the cell cycle arrest that we observed in Tsg101-deficient cells is an indirect effect due to cellular stress. Next, we performed a detailed analysis of the trafficking of EGFR as well as the morphology and function of endosomes and lysosomes using confocal microscopy. In the course of these studies, we observed a loss in co-localization between Rabaptin5 and Rab11I, which has the potential to inhibit the recycling of receptors to the cell surface. We also observed enlarged lysosomes within Tsg101-deficient cells. In subsequent studies, we were able to determine that both endocytic trafficking and degradation were accelerated in Tsg101-deficient cells. Recently, we determined that the lysosomal enlargement observed in Tsg101-deficient cells is as a result of the initiation of autophagy, a cellular process activated in response to stress. In summary, these observations suggest that Tsg101 is essential for the maintenance of individual endosomal compartments as well as a normal morphology of lysosomes. Hence, altering the expression and functionality of Tsg101 might be a strategy to modify the degradation of membrane receptors that play a role in breast cancer and other human malignancies.