Nisar Malek

Argyrin A Reveals a Critical Role for the Tumor Suppressor Protein p27kip1 in Mediating Antitumor Activities in Response to Proteasome Inhibition

A reduction in the cellular levels of the cyclin kinase inhibitor p27(kip1) is frequently found in many human cancers and correlates directly with patient prognosis. In this work, we identify argyrin A, a cyclical peptide derived from the myxobacterium Archangium gephyra, as a potent antitumoral drug. All antitumoral activities of argyrin A depend on the prevention of p27(kip1) destruction, as loss of p27(kip1) expression confers resistance to this compound. We find that argyrin A exerts its effects through a potent inhibition of the proteasome. By comparing the cellular responses exerted by argyrin A with siRNA-mediated knockdown of proteasomal subunits, we find that the biological effects of proteasome inhibition per se depend on the expression of p27(kip1).

The SCF-FBXW5 E3-ubiquitin ligase is regulated by PLK4 and targets HsSAS-6 to control centrosome duplication

Deregulated centrosome duplication can result in genetic instability and contribute to tumorigenesis. Here, we show that centrosome duplication is regulated by the activity of an E3-ubiquitin ligase that employs the F-box protein FBXW5 (ref.xa0) as its targeting subunit. Depletion of endogenous FBXW5 or overexpression of an F-box-deleted mutant version results in centrosome overduplication and formation of multipolar spindles. We identify the centriolar protein HsSAS-6 (refsxa0, ) as a critical substrate of the SCF–FBXW5 complex. FBXW5 binds HsSAS-6 and promotes its ubiquitylation in vivo. The activity of SCF–FBXW5 is in turn negatively regulated by Polo-like kinase 4 (PLK4), which phosphorylates FBXW5 at Serxa0151 to suppress its ability to ubiquitylate HsSAS-6. FBXW5 is a cell-cycle-regulated protein with expression levels peaking at the G1/S transition. We show that FBXW5 levels are controlled by the anaphase-promoting (APC/C) complex, which targets FBXW5 for degradation during mitosis and G1, thereby helping to reset the centrosome duplication machinery. In summary, we show that a cell-cycle-regulated SCF complex is regulated by the kinase PLK4, and that this in turn restricts centrosome re-duplication through degradation of the centriolar protein HsSAS-6.

C-terminal phosphorylation controls the stability and function of p27kip1.

Entry of cells into the cell division cycle requires the coordinated activation of cyclin‐dependent kinases (cdks) and the deactivation of cyclin kinase inhibitors. Degradation of p27kip1 is known to be a central component of this process as it allows controlled activation of cdk2‐associated kinase activity. Turnover of p27 at the G1/S transition is regulated through phosphorylation at T187 and subsequent SCFskp2‐dependent ubiquitylation. However, detailed analysis of this process revealed the existence of additional pathways that regulate the abundance of the protein in early G1 and as cells exit quiescence. Here, we report on a molecular mechanism that regulates p27 stability by phosphorylation at T198. Phosphorylation of p27 at T198 prevents ubiquitin‐dependent degradation of free p27. T198 phosphorylation also controls progression through the G1 phase of the cell cycle by regulating the association of p27 with cyclin–cdk complexes. Our results unveil the molecular composition of a pathway, which regulates the abundance and activity of p27kip1 during early G1. They also explain how the T187‐ and the T198‐dependent turnover systems synergize to allow cell cycle progression in G1.

Epidemiological trends in incidence and mortality of hepatobiliary cancers in Germany

Abstract Objective. While marked changes in the frequency of hepatobiliary malignancies, most notably hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), have been observed in different populations, no such data have been reported for Germany. We aimed to provide epidemiological data on recent trends in liver-related mortality, specifically mortality from hepatobiliary malignancies, in Germany. Material and methods. We used incidence and mortality data to determine changes in the frequency of malignant and non-malignant liver disease in Germany over the past 30 years. Results. While overall liver disease mortality has slightly declined in Germany, deaths from hepatobiliary malignancies have declined in women, but remained constant in men. Among hepatobiliary malignancies, ICC stands out, because mortality has more than tripled both in men and women between 1998 and 2008. This is mirrored by a marked increase in new cases reported to local cancer registries, that is, incidence. Over the same time period, HCC and extrahepatic cholangiocarcinoma (ECC) have remained largely constant while gall bladder cancers (GBC) have declined twofold. The rapid rise in ICC is in line with finding from different regions worldwide, but in contrast to recent data from Denmark and France, two of Germanys direct neighbors. Conclusions. The incidence of and mortality from ICC are rising markedly in Germany. The risk factors underlying this trend are as yet unclear.

Antitumoral Immune Response by Recruitment and Expansion of Dendritic Cells in Tumors Infected with Telomerase-Dependent Oncolytic Viruses

Virotherapy can potentially be used to induce tumor-specific immune responses and to overcome tumor-mediated tolerance mechanisms because apoptotic tumor cells are exposed together with viral danger signals during oncolysis. However, insufficient numbers of dendritic cells (DC) present at the site of oncolysis can limit a tumor-specific immune response and the resulting therapeutic benefit. We investigated MHC class I peptide-specific immune responses against model antigens ovalbumin (OVA) and hemagglutinin (HA) in mouse tumor models that support efficient replication of the oncolytic adenovirus hTert-Ad. Virotherapy resulted in peptide-specific cytotoxic T-cell responses against intracellular tumor antigens. Triggering of DC and T-cell infiltration to the oncolytic tumors by macrophage inflammatory protein 1alpha (MIP-1alpha, CCL3) and Fms-like tyrosine kinase-3 ligand (Flt3L) enhanced both antitumoral and antiviral immune responses. Although immune-mediated clearance of the virus can restrict therapeutic efficacy of virotherapy, MIP-1alpha/FLT3L-augmented hTert-Ad virotherapy inhibited local tumor growth more effectively than virotherapy alone. In agreement with the hypothesis that immune-mediated mechanisms account for improved outcome in MIP-1alpha/FLT3L virotherapy, we observed systemic antitumoral effects by MIP-1alpha/FLT3L virotherapy on uninfected lung metastasis in immunocompetent mice but not in nude mice. Furthermore, MIP-1alpha/FLT3L virotherapy of primary tumors was strongly synergistic with tumor DC vaccination in inhibition of established lung metastasis. Combined viroimmunotherapy resulted in long-term survival of 50% of treated animals. In summary, improvement of cross-presentation of tumor antigens by triggering of DC and T-cell infiltration during virotherapy enhances antitumoral immune response that facilitates an effective viroimmunotherapy of primary tumors and established metastases.

Constitutive Turnover of Cyclin E by Cul3 Maintains Quiescence

ABSTRACT Two distinct pathways for the degradation of mammalian cyclin E have previously been described. One pathway is induced by cyclin E phosphorylation and is dependent on the Cul1/Fbw7-based E3 ligase. The other pathway is dependent on the Cul3-based E3 ligase, but the mechanistic details of this pathway have yet to be elucidated. To establish the role of Cul3 in the degradation of cyclin E in vivo, we created a conditional knockout of the Cul3 gene in mice. Interestingly, the biallelic loss of Cul3 in primary fibroblasts derived from these mice results in increased cyclin E expression and reduced cell viability, paralleling the loss of Cul3 protein expression. Cell cycle analysis of viable, Cul3 hypomorphic cells shows that decreasing the levels of Cul3 increases both cyclin E protein levels and the number of cells in S phase. In order to examine the role of Cul3 in an in vivo setting, we determined the effect of deletion of the Cul3 gene in liver. This gene deletion resulted in a dramatic increase in cyclin E levels as well as an increase in cell size and ploidy. The results we report here show that the constitutive degradation pathway for cyclin E that is regulated by the Cul3-based E3 ligase is essential to maintain quiescence in mammalian cells.

p27kip1: a target for tumor therapies?

The cyclin kinase inhibitor p27kip1 acts as a potent tumor supressor protein in a variety of human cancers. Its expression levels correlate closely with the overall prognosis of the affected patient and often predict the outcome to different treatment modalities. In contrast to other tumor suppressor proteins p27 expression levels in tumor cells are frequently regulated by ubiquitin dependent proteolysis. Re-expression of p27 in cancer cells therefore does not require gene therapy but can be achieved by interfering with the protein turnover machinery. In this review we will summarize experimental results which highlight the potential use of p27 as a target for oncological therapies.

Plk2 regulates centriole duplication through phosphorylation-mediated degradation of Fbxw7 (human Cdc4)

Polo-like kinases (Plks) perform crucial functions during mitosis, cytokinesis and centriole duplication. Plk2 is activated in early G1 phase and is involved in the reproduction of centrosomes. However, the mechanisms underlying Plk2-induced centriole duplication are incompletely understood. Here, we show that Plk2 directly targets the F-box protein F-box/WD repeat-containing protein 7 (Fbxw7), which is a regulator of the ubiquitin-mediated degradation of cyclin E. Plk2 phosphorylates Fbxw7 on serine 176 and the two proteins form a complex in vitro and in vivo. Phosphorylation of Fbxw7 by Plk2 induces destabilization of the F-box protein resulting in accumulation of cyclin E and increased potential for centriole reproduction. In addition, loss of Fbxw7 in human cells leads to uncontrolled centriole duplication, highlighting the importance of Fbxw7 regulation by Plk2. These findings define a previously unknown Plk2-dependent pathway involved at the onset of S phase and in centrosome duplication.

Rapamycin delays tumor development in murine livers by inhibiting proliferation of hepatocytes with DNA damage

In this study, everolimus (RAD001) was used to determine the role of mammalian target of rapamycin (mTOR) in hepatocarcinogenesis. We show that RAD001 effectively inhibits proliferation of hepatocytes during chronic liver injury. Remarkably, the ability of RAD001 to impair cell cycle progression requires activation of the DNA damage response; loss of p53 significantly attenuates the antiproliferative effects of mTOR inhibition. RAD001 modulates the expression of specific cell cycle–related proteins and the assembly of cyclin–cyclin‐dependent kinase complexes to prevent cell cycle progression. Furthermore, RAD001 sustains the apoptosis sensitivity of hepatocytes during chronic liver injury by inhibiting p53‐induced p21 expression. Long‐term treatment with RAD001 markedly delays DNA damage–induced liver tumor development. Conclusion: We provide evidence that mTOR inhibition has a substantial effect on sequential carcinogenesis and may offer an effective strategy to delay liver tumor development in patients at risk. (HEPATOLOGY 2009;50:500–509.)

Nuclear expression of the ubiquitin ligase seven in absentia homolog (SIAH)-1 induces proliferation and migration of liver cancer cells

BACKGROUND & AIMSnDifferential expression of tumor-relevant proteins based on aberrant proteasomal degradation may contribute to human (hepato)carcinogenesis. Recently, we identified the E3 ubiquitin ligase seven in absentia homolog (SIAH)-1 as frequently dysregulated in human hepatocellular carcinoma (HCC). We therefore systematically analyzed the expression, functional relevance, as well as possible downstream effectors of SIAH-1 in human liver carcinogenesis.nnnMETHODSnSIAH-1 expression was analyzed at the transcript and protein levels in human hepatocarcinogenesis and in HCC cells. Proliferation, apoptosis, and migration of different HCC cell lines were examined after siRNA-mediated inhibition of SIAH-1. In order to identify downstream effectors that mediate SIAH-1 effects, correlative analyses of protein expression profiles were performed.nnnRESULTSnIn HCC tissues both reduction of cytoplasmic SIAH-1 and especially its nuclear accumulation positively correlated with HCC progression. RNA interference revealed that nuclear expression of SIAH-1 predominantly supported HCC cell proliferation and migration while only moderately affecting anti-apoptosis. In de-differentiated human HCCs, nuclear SIAH-1 accumulation significantly correlated with the expression of the transcription factor far-upstream element (FUSE)-binding protein (FBP)-3. In vitro, SIAH-1 positively and indirectly regulated FBP-3 which itself primarily supported HCC cell proliferation. Indeed, high level expression of FBP-3 in human HCCs significantly correlated with reduced overall survival of patients.nnnCONCLUSIONSnNuclear accumulation of the E3 ubiquitin ligase SIAH-1 supports different pro-tumorigenic cellular processes associated with tumor growth and tumor cell dissemination in human hepatocarcinogenesis. It promotes HCC cell proliferation by at least partly employing the transcription factor FBP-3. Therefore, interference with SIAH-1 activity represents a promising approach to suppress HCC growth.