Activation of Cell Cycle Arrest and Apoptosis by the Proto-Oncogene Pim-2

Potent survival effects have been ascribed to the serine/threonine kinase proto-oncogene PIM-2. Elevated levels of PIM-2 are associated with various malignancies. In human cells, a single Pim-2 transcript gives rise mainly to two protein isoforms (34, 41 kDa) that share an identical catalytic site but differ at their N-terminus, due to in-frame alternative translation initiation sites. In this study we observed that the 34 kDa PIM-2 isoform has differential nuclear and cytoplasmic forms in all tested cell lines, suggesting a possible role for the balance between these forms for PIM-2's function. To further study the cellular role of the 34 kDa isoform of PIM-2, an N-terminally HA-tagged form of this isoform was transiently expressed in HeLa cells. Surprisingly, this resulted in increased level of G1 arrested cells, as well as of apoptotic cells. These effects could not be obtained by a Flag-tagged form of the 41 kDa isoform. The G1 arrest and apoptotic effects were associated with an increase in T14/Y15 phosphorylation of CDK2 and proteasom-dependent down-regulation of CDC25A, as well as with up-regulation of p57, E2F-1, and p73. No such effects were obtained upon over-expression of a kinase-dead form of the HA-tagged 34 kDa PIM-2. By either using a dominant negative form of p73, or by over-expressing the 34 kDa PIM-2 in p73-silenced cells, we demonstrated that these effects were p73-dependent. These results demonstrate that while PIM-2 can function as a potent survival factor, it can, under certain circumstances, exhibit pro-apoptotic effects as well

p57 expression and T14/Y15 phosphorylation on CDK2 in HeLa cells over-expressing the 34 kDa PIM-2 isoform.

<p>(<b>A</b>) RT-PCR analysis of p57 transcripts in control cells (HA control) and in Pim2 over-expressing cells (HA Pim-2). Actin specific primers were used as reference for equal loading. Western blot analysis was used to evaluate the p57 protein in nuclear extracts (40 µg) from HA control cells and from cells over-expressing Pim-2 (HA Pim-2). Anti-RCC1 antibodies were used as reference for equal loading. The average relative level of p57 in Pim-2 over-expressing cells (Pim-2) compared to HA control cells (HA) is depicted in the right panel. Comparison was based on densitometric analysis of p57 signals normalized according to the RCC1 signal (average of three independent experiments). Average level of p57 in control cells was determined as 1. Differences were just above the statistically significance of p<0.05. (<b>B</b>) Western blot analysis of phosphorylated CDK2 on T14/Y15 (pCDK2) in nuclear extracts from cells over-expressing Pim-2 (HA Pim-2) and HA control cells, using phospho T14/Y15 specific antibodies. Blots were stripped and reprobed once with CDK2 specific antibodies and then once again with RCC1 antibodies as reference for equal loading. The average relative level of pCDK2 in Pim-2 over-expressing cells (Pim-2) compared to HA control cells (HA) is depicted in the right panel. Comparison was based on densitometric analysis of pCDK2 signals normalized according to the RCC1 signal (average of three independent experiments). Average level of pCDK2 in control cells was determined as 1. Asterisk represents statistically significant differences (p<0.04) (<b>C</b>) Western blot analysis of pCDK2 in total protein extracts (40 µg) from Pim-2 silenced cells, via siRNA (si-Pim), and from cells transfected with scrambled control siRNAs (si-control). Antibodies specific to β-actin were used as reference for equal loading.</p

PIM-2's overexpression in HeLa cells promotes G1 arrest and increases apoptosis.

<p>(<b>A</b>) Upper panel - Light microscope images (×40) of cells 48 hours after transfecting equal amounts of cells with either HA-Pim-2 vector (pHA-Pim2) or empty control vector (pHA-empty), and under identical culture conditions. Middle panel – Western blot analysis, using anti HA antibodies, showing the relative amounts of either HA-PIM-2 or HA-PIM-2 KD in the tested cells. Lower panel - Fold increase in cell number calculated 48 hours after transfecting equal amounts of cells with either the HA-Pim2 plasmid (Pim-2), an empty HA control vector (HA) or with a kinase dead form of Pim-2 (PimKD). Data shown are the average of four independent experiments. Asterisk represents statistically significant differences (p<0.05). (<b>B</b>) FACS analysis of cell cycle distribution of PI stained cells 48 hours after transfection with either HA-Pim2 plasmid (Pim-2), HA-Pim2 kinase-dead plasmid (Pim-2 KD), control empty vector (HA) or untreated cells (HeLa). (<b>C</b>) A comparison between the average percentage of cells (four independent experiments) at the sub-G1 and G1 phases, in Pim-2 expressing cells versus HA control cells (Asterisks represents statistically significant differences p<0.014 and p<0.007, respectively).</p

Down-regulation of CDC25A expression in HeLa cells over-expressing the 34 kDa form of PIM-2.

<p>(<b>A</b>) Western blot analysis of the CDC25A protein in nuclear extracts (40 µg) from cells over-expressing Pim-2 (Pim-2) and control cells transfected with empty HA vector (HA). Anti-RCC1 antibodies were used as reference for equal loading. The average relative level of CDC25A in Pim-2 over-expressing cells (Pim-2) compared to HA control cells (HA) is depicted in the right panel. Comparison was based on densitometric analysis of CDC25A signals normalized according to the RCC1 signal (average of three independent experiments). Average level of CDC25A in control cells was determined as 1. Asterisk represents statistically significant differences (p<0.05). (<b>B</b>) RT-PCR analysis of Cdc25A transcripts in Pim2 over-expressing cells (Pim-2) and in control cells (HA). Actin specific primers were used as reference for equal loading. (<b>C</b>) <b>PIM-2 promotes CDC25A degradation via the proteasome.</b> Western blot analyses of β-catenin (top panel), as control for proteasomal inhibition, and CDC25A (botom panel), were performed on total protein extracts (40 µg) from cells over-expressing Pim-2 (Pim-2) and control cells (HA) after treatment with the proteasome inhibitor, MG-132, for 0, 2 or 5 hours (h). Antibodies specific to tubulin were used as a reference for equal loading. (<b>D</b>) <b>PIM-2 directly phosphorylates CDC25A in an </b><b><i>in-vitro</i></b><b> kinase assay.</b> 293 cells were transfected (+), or not (−), with a Flag-tagged CDC25A expressing vector, and the tagged protein was immunoprecipitated using anti Flag antibodies. The immunoprecipitated protein was used as a substrate in a PIM-2 kinase assay (lower panel). Expression of the tagged protein was verified by Western analysis (upper panel), and equal loading was verified by stripping the blot and probing it with anti tubulin antibodies (middle panel).</p

Dominant negative(DN) p73 or p73 silencing reverse the PIM-2 effect on cells over-expressing the 34 kDa PIM-2 isoform.

<p>(<b>A</b>) Light microscopic view (×40) of cells 48 hours after transfecting equal amounts of cells with an empty HA vector as control (control), HA-Pim2 plasmid alone (Pim-2) and a HA-Pim-2 plasmid together with a dominant negative form of p73 (Pim-2+p73DN), and under identical culture conditions. (<b>B</b>) Percent of sub-G1 apoptotic cells, and (<b>C</b>) percent of cells in the G1 phase of the cell cycle, in the specified cultures, as revealed by FACS analysis. Results represent an average of four independent experiments. Asterisk represents statistical significance p<0.01. (<b>D</b>) Western analysis showing siRNA-silencing of p73 compared to untreated control or control of cell treated with scrambled siRNA. (<b>E</b>) FACS analysis of cell cycle pattern of PI stained cells to which siRNAs were introduced [control scrambled (scr.) or p73-directed siRNA (p73)] 24 h prior to transfection with either empty HA plasmid (HA+scr. or HA+73) or HA-Pim-2 plasmid (PIM-2+scr. or PIM-2+73). FACS analysis was performed 76 h after transfection with plasmids. Horizontal line in each pattern indicates the channels that were included in calculation of the sub-G1 phase. (<b>F</b>) Percent of cells at the sub-G1 phase calculated from the cell cycle patterns presented in panel E.</p

A proposed model explaining the molecular pathway through which PIM-2 exerts G1-arrest and promotes apoptosis.

<p>A proposed model explaining the molecular pathway through which PIM-2 exerts G1-arrest and promotes apoptosis.</p

Over-expression of the 34 kDa isoform of the human PIM-2 protein in HeLa cells.

<p>(<b>A</b>) Nuclear and cytoplasmic protein extracts (50 µg) from cells over-expressing HA-PIM-2 (Pim-2), control cells transfected with a HA vector (HA) or untreated control cells (HeLa), were analyzed by Western blotting using antibodies against either the PIM-2 protein or the HA-tag. For equal loading reference, the blot was stripped and re-probed with antibodies against β-actin (cytoplasmic fractions) or RCC1 (nuclear fractions). (<b>B</b>) Immunocytochemical analysis of HeLa cells transiently transfected with the HA-Pim-2 encoding plasmid, using either rabbit anti-PIM-2 antibodies (PIM-2) or mouse anti-HA antibodies (HA). Alexa 488 conjugated anti-rabbit (green) and Alexa 594 conjugated anti-mouse (red) secondary antibodies were used for PIM-2 and HA detection, respectively. In right lower panel PIM-2 and HA signals are merged. Nuclei were stained with Dapi (blue). Bar represents 10 µm. (<b>C</b>) Total protein extracts (50 µg) from cells over-expressing the 34 kDa isoform of PIM-2 tagged with the HA-tag at its N-terminus and with the Myc-His-tag at its C-terminus (HA-PIM-2-Myc), or from untreated control cells (HeLa), were analyzed by Western blotting, using antibodies against the PIM-2 protein (PIM-2). The blot was stripped and re-probed with antibodies against the Myc-tag (Myc), stripped again and re-probed with antibodies against the HA-tag. For equal loading reference, the blot was stripped once again and re-probed with antibodies against tubulin.</p

Hepatitis C Virus Enhances the Invasiveness of Hepatocellular Carcinoma via EGFR-Mediated Invadopodia Formation and Activation

Hepatocellular carcinoma (HCC) represents the fifth most common cancer worldwide and the third cause of cancer-related mortality. Hepatitis C virus (HCV) is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis, and eventually HCC. HCV is the most common risk factor for HCC in western countries and leads to a more aggressive and invasive disease with poorer patient survival rates. However, the mechanism by which the virus induces the metastatic spread of HCC tumor cells through the regulation of invadopodia, the key features of invasive cancer, is still unknown. Here, the integration of transcriptome with functional kinome screen revealed that HCV infection induced invasion and invadopodia-related gene expression combined with activation of host cell tyrosine kinases, leading to invadopodia formation and maturation and consequent cell invasiveness in vitro and in vivo. The promotion of invadopodia following HCV infection was mediated by the sustained stimulation of epidermal growth factor receptor (EGFR) via the viral NS3/4A protease that inactivates the T-cell protein tyrosine phosphatase (TC-PTP), which inhibits EGFR signaling. Characterization of an invadopodia-associated gene signature in HCV-mediated HCC tumors correlated with the invasiveness of HCC and poor patient prognosis. These findings might lead to new prognostic and therapeutic strategies for virus-mediated invasive cancer

Dysregulation of the cohesin subunit RAD21 by Hepatitis C virus mediates host-virus interactions

Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV possesses an RNA genome and its replication is confined to the cytoplasm. Yet, infection with HCV leads to global changes in gene expression, and chromosomal instability (CIN) in the host cell. The mechanisms by which the cytoplasmic virus affects these nuclear processes are elusive. Here, we show that HCV modulates the function of the Structural Maintenance of Chromosome (SMC) protein complex, cohesin, which tethers remote regions of chromatin. We demonstrate that infection of hepatoma cells with HCV leads to up regulation of the expression of the RAD21 cohesin subunit and changes cohesin residency on the chromatin. These changes regulate the expression of genes associated with virus-induced pathways. Furthermore, siRNA downregulation of viral-induced RAD21 reduces HCV infection. During mitosis, HCV infection induces hypercondensation of chromosomes and the appearance of multi-centrosomes. We provide evidence that the underlying mechanism involves the viral NS3/4 protease and the cohesin regulator, WAPL. Altogether, our results provide the first evidence that HCV induces changes in gene expression and chromosome structure of infected cells by modulating cohesin