HS1-associated protein X-1 regulates carcinoma cell migration and invasion via clathrin-mediated endocytosis of integrin alphavbeta6.

Enhanced expression levels of integrin alphavbeta6 have been linked to more aggressive invasive carcinoma cell behavior and poorer clinical prognosis. However, how alphavbeta6 determines invasion and the dynamics of integrin alphavbeta6 regulation in tumor cells are poorly understood. We have identified the 35-kDa HS1-associated protein X-1 (HAX-1) protein as a novel binding partner of the beta6 cytoplasmic tail using a yeast two-hybrid screen. We show that alphavbeta6-dependent migration is blocked following small interfering RNA (siRNA)-mediated depletion of HAX-1 in oral squamous cell carcinoma cell lines. Using both siRNA and membrane-permeable peptides, we show that alphavbeta6-dependent migration and invasion require HAX-1 to bind directly to beta6 and thereby regulate clathrin-mediated endocytosis of alphavbeta6 integrins. Progression of oral cancer is associated with enhanced expression of alphavbeta6 and HAX-1 proteins in patient tissue. This report establishes that integrin endocytosis is required for alphavbeta6-dependent carcinoma cell motility and invasion and suggests that this process is an important mechanism in cancer progression.

Inducible expression of a dominant negative DNA polymerase-gamma depletes mitochondrial DNA and produces a rho0 phenotype.

We report the inducible, stable expression of a dominant negative form of mitochondria-specific DNA polymerase-gamma to eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA polymerase-gamma harboring a D1135A substitution (POLGdn). The cells rapidly lost mtDNA (t1/2 = 2-3 days) when expression of the transgene was induced. Concurrent reduction of mitochondrial encoded mRNA and protein, decreased cellular growth rate, and compromised respiration and mitochondrial membrane potential were observed. mtDNA depletion was reversible, as demonstrated by restoration of mtDNA copy number to normal within 10 days when the expression of POLGdn was suppressed following a 3-day induction period. Long term (20 days) expression of POLGdn completely eliminated mtDNA from the cells, resulting in rho0 cells that were respiration-deficient, lacked electron transport complex activities, and were auxotrophic for pyruvate and uridine. Fusion of the rho0 cells with human platelets yielded clonal cybrid cell lines that were populated exclusively with donor-derived mtDNA. Respiratory function, mitochondrial membrane potential, and electron transport activities were restored to normal in the cybrid cells. Inducible expression of a dominant negative DNA polymerase-gamma can yield mtDNA-deficient cell lines, which can be used to study the impact of specific mtDNA mutations on cellular physiology, and to investigate mitochondrial genome function and regulation.