Melanoma, a malignant neoplasm of melanocytes, is the most lethal form of skin cancer. A majority of melanomas are driven by activating mutations in the kinase BRAF, which drives cellular proliferation through constitutive stimulation of the mitogen-activated protein kinase (MAPK) signaling pathway. Intriguingly, expression of oncogenic BRAF alone in vivo is insufficient to promote melanoma; rather, its expression leads to the development of benign nevi (moles) comprised of growth-arrested melanocytes. The acquisition of additional genetic or epigenetic changes is therefore critical for melanocytes to evade arrest and drive melanomagenesis, however the identity of these changes remains incompletely understood. Here we demonstrate that expression of oncogenic BRAF leads to activation of the Hippo tumor suppressor pathway in vitro, which acts to limit melanocyte proliferation through the inhibition of the pro-growth transcriptional co-activators YAP and TAZ. Melanocyte-specific inactivation of Hippo signaling in vivo, via deletion of the Hippo kinases Lats1/2 alone, or in conjunction with oncogenic Braf expression, potently induces melanoma development in mice. Collectively, our data reveal that the Hippo tumor suppressor pathway represents an important barrier to melanoma development, and implicates YAP and TAZ as new therapeutic targets for the treatment of human melanoma.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

Hereditary spastic paraplegia type 54 is a rare autosomal recessive neurological gait disorder characterized by paraplegia, muscle spasticity, and intellectual disability. This length-dependent distal axonopathy is caused by mutations in the DDHD2 gene, which encodes the intracellular phospholipase A1 DDHD2. Little is known about the molecular function of the DDHD2 protein, especially in the context of HSP54. Thus, there is a need to further investigate its molecular functions and investigate the impact of DDHD2 deficiency in disease-relevant cells. Here, lipidomic profiling of dermal fibroblasts derived from three unrelated patients has revealed 19 glycerophosphoethanolamine species at differential levels in patients relative to unaffected controls. However, patient cells appear to have an unaffected Golgi apparatus morphology and lipid droplet formation, despite DDHD2’s proposed roles in these processes. To study the gene function in neuronal cells, I transdifferentiated the fibroblasts into induced neuronal precursor cells and found all the patient cells arrested in the G0/G1 phase of upon conversion. Given that these cell lines are unsustainable, I generated a stable knockdown cell line in the highly proliferative HEK293A to study the molecular biology of DDHD2. The knockdown cells had a reduced growth, were delayed in the G2/M phase of the cell cycle, and became multinucleated. I then treated the cells with antineoplastic compounds paclitaxel and nocodazole and found more knockdown cells in G0/G1 than controls, suggesting the possible occurrence of mitotic slippage. Lastly, I report a novel subcellular localization for DDHD2 at the microtubule organization center.