Faculty Bibliography

ABSTRACT: Angiogenesis is essential for promoting growth and metastasis of solid tumors by ensuring blood supply to the tumor mass. Targeting angiogenesis is therefore an attractive approach to therapeutic intervention of cancer. Tumor angiogenesis is a process that is controlled by a complex network of molecular components including sensors, signaling transducers, and effectors, leading to cellular responses under hypoxic conditions. Positioned at the center of this network are the hypoxia-inducible factors (HIFs). HIF-1 is a major transcription factor that consists of two subunits, HIF-1alpha and HIF-1beta. It mediates transcription of a spectrum of gene targets whose products are essential for mounting hypoxic responses. HIF-1alpha protein level is very low in the normoxic condition but is rapidly elevated under hypoxia. This dramatic change in the cellular HIF-1alpha level is primarily regulated through the proteosome-mediated degradation process. In the past few years, scientific progress has clearly demonstrated that HIF-1alpha phosphorylation is mediated by several families of protein kinases including GSK3beta and ERKs both of which play crucial roles in the regulation of HIF-1alpha stability. Recent research progress has identified that Polo-like kinase 3 (Plk3) phosphorylates HIF-1alpha at two previously unidentified serine residues and that the Plk3-mediated phosphorylation of these residues results in destabilization of HIF-1alpha. Plk3 has also recently been found to phosphorylate and stabilize PTEN phosphatase, a known regulator of HIF-1alpha and tumor angiogenesis. Given the success of targeting protein kinases and tumor angiogenesis in anti-cancer therapies, Plk3 could be a potential molecular target for the development of novel and effective therapeutic agents for cancer treatment.

SALL4 is a transcription factor that plays a key role in the maintenance and self-renewal of embryonic stem cells and hematopoietic stem cells. Given that little is known about regulation of SALL4, we studied biochemical modifications of SALL4B, a major splicing variant of SALL4, and elucidated their biological function. SALL4B was primarily modified by ubiquitination when it was expressed in both Sf9 and HEK293T cells. A significant fraction of SALL4B was further modified by sumoylation when it was expressed in HEK293T cells. Constitutive SUMO-modification of SALL4B was also detected in Tera-1, a cell line of the teratocarcinoma origin. SALL4B sumoylation was independent of ubiquitination and lysine residues 156, 316, 374, and 401 were essential for sumoylation. Chromatin fraction contained more SUMO-deficient SALL4B. Despite a shorter half-life than the wild-type counterpart, SUMO-deficient SALL4B interacted with OCT4 more efficiently than the wild-type SALL4B. RNAi-mediated silencing of SALL4 expression caused significant down-regulation of both OCT4 and SOX2, which was rescued by ectopic expression of SALL4B but not by SUMO-deficient mutant. Significantly, compared with the wild-type SALL4B, SUMO-deficient mutant exhibited compromised trans-activation or trans-repression activities in reporter gene assays. Combined, our studies reveal sumoylation as a novel form of post-translational modification for regulating the stability, subcellular localization, and transcriptional activity of SALL4.

Accurate chromosome segregation during mitosis and meiosis is regulated and secured by several distinctly different yet intricately connected regulatory mechanisms. As chromosomal instability is a hallmark of a majority of tumors as well as a cause of infertility for germ cells, extensive research in the past has focused on the identification and characterization of molecular components that are crucial for faithful chromosome segregation during cell division. Shugoshins, including Sgo1 and Sgo2, are evolutionarily conserved proteins that function to protect sister chromatid cohesion, thus ensuring chromosomal stability during mitosis and meiosis in eukaryotes. Recent studies reveal that Shugoshins in higher animals play an essential role not only in protecting centromeric cohesion of sister chromatids and assisting bi-orientation attachment at the kinetochores, but also in safeguarding centriole cohesion/engagement during early mitosis. Many molecular components have been identified that play essential roles in modulating/mediating Sgo functions. This review primarily summarizes recent advances on the mechanisms of action of Shugoshins in suppressing chromosomal instability during nuclear division in eukaryotic organisms.

Chromosome instability (CIN) is found in 85% of colorectal cancers. Defects in mitotic processes are implicated in high CIN and may be critical events in colorectal tumorigenesis. Shugoshin-1 (SGO1) aids in the maintenance of chromosome cohesion and prevents premature chromosome separation and CIN. In addition, integrity of the centrosome may be compromised due to the deficiency of Cohesin and Sgo1 through the disengagement of centrioles. We report here the generation and characterization of SGO1-mutant mice and show that haploinsufficiency of SGO1 leads to enhanced colonic tumorigenesis. Complete disruption of SGO1 results in embryonic lethality, whereas SGO1 (+/-) mice are viable and fertile. Haploinsufficiency of SGO1 results in genomic instability manifested as missegregation of chromosomes and formation of extra centrosomal foci in both murine embryonic fibroblasts and adult bone marrow cells. Enhanced CIN observed in SGO1-deficient mice resulted in an increase in formation of aberrant crypt foci (ACF) and accelerated development of tumors after exposure to azoxymethane (AOM), a colon carcinogen. Together, these results suggest that haploinsufficiency of SGO1 causes enhanced CIN, colonic preneoplastic lesions and tumorigenesis in mice. SGO1 is essential for the suppression of CIN and tumor formation.

BubR1 is an important component of the spindle assembly checkpoint, and deregulated BubR1 functions frequently result in chromosomal instability and malignant transformation. We recently demonstrated that BubR1 was modified by sumoylation, and that lysine 250 (K250) functions as the crucial site for this modification. BubR1 sumoylation was neither required for its activation nor for binding to kinetochores. However, ectopically expressed sumoylation-deficient BubR1 mutants were retained on the kintochores even after apparent chromosome congression. The kinetochore retention of the sumoylation-deficient mutant of BubR1 caused an anaphase delay coupled with premature sister chromatid separation. Moreover, BubR1 interacted with unphosphorylated Sgo1, and its sumoylation facilitated the interaction. BubR1 sumoylation was inversely associated with its acetylation during mitotic progression. Trichostatin A, a protein deacetylase inhibitor, significantly compromised BubR1 sumoylation. Combined, these results reveal that BubR1 sumoylation plays an important role in its timely removal from the kinetochores and the checkpoint inactivation, thus allowing normal anaphase entry and chromosome segregation.

In order to maintain chromosomal stability during cell division, eukaryotic cells have evolved a number of surveillance mechanisms termed checkpoints. These checkpoints monitor the completion of essential molecular and cellular processes of one stage before entering another. The spindle checkpoint watches the bi-orientation attachment of spindle microtubules to all condensed chromosomes before initiation of nuclear division during mitosis. Histones are subject to a number of post-translational modifications during the cell cycle, which may in turn modify or facilitate cell cycle progression. Recent studies suggest that mitotic proteins including Bub1 and Sgo1 that are involved in the spindle checkpoint also play a major role in the regulation of histone modifications and chromatin remodeling. This mini-review summarizes emerging information about the new role of spindle checkpoint proteins in chromatin remodeling.

BubR1 functions as a crucial component that monitors proper chromosome congression and mitotic timing during cell division. We investigated molecular regulation of BubR1 and found that BubR1 was modified by an unknown post-translation mechanism during the cell cycle, resulting in a significant mobility shift on denaturing gels. We termed it BubR1-M as the nature of modification was not characterized. Extended (>24 h) treatment of HeLa cells with a microtubule disrupting agent including nocodazole and taxol or release of mitotic shake-off cells into fresh medium induced BubR1-M. BubR1-M was derived from neither phosphorylation nor acetylation. Ectopic expression coupled with pulling down analyses showed that BubR1-M was derived from SUMO modification. Mutation analysis revealed that lysine 250 was a crucial site for sumoylation. Significantly, compared with the wild-type control, ectopic expression of a sumoylation-deficient mutant of BubR1 induced chromosomal missegregation and mitotic delay. Combined, our study identifies a new type of post-translational modification that is essential for BubR1 function during mitosis.

Elevated extracellular solute concentration (hyperosmotic stress) perturbs cell function and stimulates cell responses by evoking MAPK cascades and activating AP-1 transcription complex resulting in alterations of gene expression, cell cycle arrest, and apoptosis. The results presented here demonstrate that hyperosmotic stress elicited increases in ATF-2 phosphorylation through a novel Polo-like kinase 3 (Plk3) pathway in human corneal epithelial (HCE) cells. We found in hyperosmotic stress-induced HCE cells that Plk3 transferred to the nuclear compartment and was colocalized with ATF-2 in nuclei. Kinase activity of Plk3 was significantly activated by hyperosmotic stimulation. Further downstream, active Plk3 phosphorylated ATF-2 at the Thr-71 site in vivo and in vitro. Overexpression of Plk3 and its mutants enhanced hyperosmotic stress-induced ATF-2 phosphorylation. In contrast, suppression of Plk3 by knocking down Plk3 mRNA effectively diminished the effect of hyperosmotic stress-induced ATF-2 phosphorylation. The effect of hyperosmotic stress-induced activation of Plk3 on ATF-2 transcription factor function was also examined in CRE reporter-overexpressed HCE cells. Our results for the first time reveal that hyperosmotic stress can activate the Plk3 signaling pathway that subsequently regulates the AP-1 complex by directly phosphorylating ATF-2 independent from the effects of JNK and p38 activation

Disruption of cell cycle checkpoints and interference with the normal cell cycle progression frequently result in cell death or malignant transformation. Hexavalent chromium [Cr(VI)] is a well-known carcinogen that has been implicated in the occurrence of many types of human malignancies, including lung cancer. However, the exact mechanism by which Cr(VI) causes malignant transformation in the lung remains unknown. We have demonstrated that chronic exposure to a non-cytotoxic concentration of Cr(VI) induced a variety of chromosomal abnormalities, including premature sister chromatid separation, chromosomal breakage and the presence of lagging/misaligned chromosomes. After treatment with nocodazole, both HeLa and normal lung bronchial epithelial cells were arrested at mitosis. However, Cr(VI) significantly compromised M-phase arrest induced by nocodazole. Cr(VI) suppressed BubR1 activation and reduced expression of Emi1, leading to an unscheduled activation of APC/C. Consistent with this observation, Cr(VI) treatment caused enhanced polyubiquitination of geminin during mitotic release, while it deregulated the activity of Cdt1, a DNA replication licensing factor. Combined, these results suggest that Cr(VI)-induced chromosomal instability is partly due to a perturbation of APC/C activities, leading to chromosomal instability

PURPOSE: Hyperosmotic stress causes cell shrinkage, perturbs cell function, and damages DNA, resulting in cell cycle arrest and apoptosis. In the present study, the authors explore the mechanism involving hyperosmotic stress-induced activation of c-Jun/AP-1 through a novel Plk3 pathway in human corneal epithelial cells. METHODS: Human primary corneal epithelial cells and cell line were cultured in a serum-free keratinocyte medium and DMEM/F12 medium containing 10% FBS in a 37 degrees C incubator supplied with 5% CO(2), respectively. Western blot analysis was used to determine protein expression and phosphorylation levels. Protein kinase activities were measured by immunocomplex kinase assay. Cell viability and apoptosis were determined by MTT assay and caspase-3 (DEVDase) activity. RESULTS: It was found that hyperosmotic stress-induced increases in the phosphorylation of c-Jun, resulting in apoptosis through the activation of Plk3 in human corneal epithelial cells. Plk3 was activated by extracellular hyperosmotic stress to directly phosphorylate c-Jun in the serine 63 and 73 residues. Hyperosmotic stress-induced c-Jun phosphorylation was enhanced by overexpression of constitutively positive Plk3 mutants and suppressed by the knockdown of Plk3 mRNA with Plk3-specific siRNA. Further studies indicated that the phosphorylation of c-Jun by Plk3 was responsible for hyperosmotic stress-induced apoptosis, which was independent from activation of the JNK signaling pathway in human corneal epithelial cells. CONCLUSIONS: These results, for the first time, provide a novel and alternative signaling mechanism that involves hyperosmotic stress-induced activation of the Plk3 pathway in addition to JNK/p38 MAPK pathways to regulate the c-Jun/AP-1 transcriptional complex and human corneal epithelial cell fate