Chioniso Patience Masamha

Cyclin D1 Degradation Is Sufficient to Induce G1 Cell Cycle Arrest despite Constitutive Expression of Cyclin E2 in Ovarian Cancer Cells

D- and E-type cyclins mediate G(1)-S phase cell cycle progression through activation of specific cyclin-dependent kinases (cdk) that phosphorylate the retinoblastoma protein (pRb), thereby alleviating repression of E2F-DP transactivation of S-phase genes. Cyclin D1 is often overexpressed in a variety of cancers and is associated with tumorigenesis and metastasis. Loss of cyclin D can cause G(1) arrest in some cells, but in other cellular contexts, the downstream cyclin E protein can substitute for cyclin D and facilitate G(1)-S progression. The objective of this study was to determine if a flexible heteroarotinoid anticancer compound, SHetA2, regulates cell cycle proteins and cell cycle progression in ovarian cancer cells. SHetA2 induced cyclin D1 phosphorylation, ubiquitination, and proteasomal degradation, causing G(1) arrest in ovarian cancer cells despite continued cyclin E2 expression and independently of p53 and glycogen synthase kinase-3beta. Cyclin D1 loss inhibited pRb S780 phosphorylation by cyclin D1-cdk4/6 and released p21 from cyclin D1-cdk4/6-p21 protein complexes to form cyclin E2-cdk2-p21 complexes, which repressed phosphorylation of pRb S612 by cyclin E2-cdk2 and ultimately E2F-DP transcriptional activity. G(1) arrest was prevented by overexpression or preventing degradation of cyclin D1 but not by restoration of pRb S612 phosphorylation through p21 knockdown. In conclusion, we show that loss of cyclin D1 in ovarian cancer cells treated with SHetA2 is sufficient to induce G(1) cell cycle arrest and this strategy is not impeded by the presence of cyclin E2. Therefore, cyclin D1 is a sufficient therapeutic target in ovarian cancer cells.

CFIm25 links alternative polyadenylation to glioblastoma tumour suppression

The global shortening of messenger RNAs through alternative polyadenylation (APA) that occurs during enhanced cellular proliferation represents an important, yet poorly understood mechanism of regulated gene expression. The 3′ untranslated region (UTR) truncation of growth-promoting mRNA transcripts that relieves intrinsic microRNA- and AU-rich-element-mediated repression has been observed to correlate with cellular transformation; however, the importance to tumorigenicity of RNA 3′-end-processing factors that potentially govern APA is unknown. Here we identify CFIm25 as a broad repressor of proximal poly(A) site usage that, when depleted, increases cell proliferation. Applying a regression model on standard RNA-sequencing data for novel APA events, we identified at least 1,450 genes with shortened 3′ UTRs after CFIm25 knockdown, representing 11% of significantly expressed mRNAs in human cells. Marked increases in the expression of several known oncogenes, including cyclin D1, are observed as a consequence of CFIm25 depletion. Importantly, we identified a subset of CFIm25-regulated APA genes with shortened 3′ UTRs in glioblastoma tumours that have reduced CFIm25 expression. Downregulation of CFIm25 expression in glioblastoma cells enhances their tumorigenic properties and increases tumour size, whereas CFIm25 overexpression reduces these properties and inhibits tumour growth. These findings identify a pivotal role of CFIm25 in governing APA and reveal a previously unknown connection between CFIm25 and glioblastoma tumorigenicity.

The pro-survival function of Akt kinase can be overridden or altered to contribute to induction of apoptosis

The serine/threonine protein kinase B (PKB), which is now called Akt, has well-documented oncogenic potential and pro-survival activities that can counteract apoptosis induced by anti-cancer drugs. The goal of this review is to discuss current evidence that the pro-survival function of Akt can be overridden or converted to a pro-apoptotic function. A brief description of how upstream regulators and downstream effectors of the Akt kinase participate in a network of protection against cell death is presented. This background provides a basis for understanding how specific chemotherapeutic agents and cellular conditions can overcome the Akt pro-survival signal or alter Akt signaling in a way that converts Akt kinase activity to be directly involved in the induction of apoptosis. This pro-apoptotic activity only occurs under specific cellular conditions, since Akt can function as both a survival factor and an apoptotic factor within the same cell type. In some situations, the Akt pro-survival activity was eventually overwhelmed by prolonged treatment with chemotherapeutic agents, or was converted to a pro-apoptotic function upon prolonged hyperactivation of the Akt kinase activity, or by nuclear retention or unbalanced phosphorylation of the Akt protein. Increased levels of intracellular oxidation stimulated Akt activity and were increased by oxidative metabolism resulting from chronic Akt hyperactivity. Downstream effects on mTOR, FoxO3 transcription factors and cdk-2 affected the switch between pro-survival and pro-apoptotic functions through complex positive- and negative-feedback interactions. Upstream, caveolin-1 stimulated the pro-apoptotic function. Implications of the opposing functions of Akt in cancer therapy are discussed.

Development of flexible-heteroarotinoids for kidney cancer

Potential chemopreventive and therapeutic value of the lead Flexible Heteroarotinoid (Flex-Het), SHetA2, was indicated by growth inhibition of multiple cancer cell lines. The objective of this study was to evaluate the SHetA2 mechanism and in vivo activity in kidney cancer. SHetA2 induced apoptosis in the Caki-1 kidney cancer cell line through reduction of Bcl-2 protein and induction of PARP-1 and caspase 3 cleavages, whereas normal kidney epithelial cells exhibited resistance. Both normal and cancerous cells underwent G1 arrest and loss of Cyclin D1. Tubule differentiation was induced in organotypic cultures and xenograft tumors in association with increases in E-Cadherin mRNA and protein expression. SHetA2 repressed activity of nuclear factor-κB, a transcription factor that regulates apoptosis, Bcl-2, growth, Cyclin D1, differentiation, and E-Cadherin in the opposite manner as SHetA2. Glutathione binding and generation of reactive oxygen species were not required for these activities. Oral SHetA2 inhibited growth in one of two renal cancer xenograft models without causing mortality or weight loss. Structure function analysis of related Flex-Hets for potential improvement of SHetA2 pharmaceutical properties showed that compounds with increased hydrophilicity slightly reduced the growth inhibition efficacy, but retained the differential effect on cancer over normal cells. Flex-Hets and metabolites were not mutagenic in the Ames test. In conclusion, SHetA2 regulates growth, differentiation, and apoptosis in kidney cancer cells through multiple molecular events downstream of nuclear factor-κB repression. Increasing the hydrophilicity of Flex-Hets does not attenuate the differential effect on cancer cells over normal cells, thus offering alternatives for improvement of therapeutic value.[Mol Cancer Ther 2009;8(5):OF1–12]

3′ UTR shortening represses tumor-suppressor genes in trans by disrupting ceRNA crosstalk

Widespread mRNA 3′ UTR shortening through alternative polyadenylation1 promotes tumor growth in vivo2. A prevailing hypothesis is that it induces proto-oncogene expression in cis through escaping microRNA-mediated repression. Here we report a surprising enrichment of 3′UTR shortening among transcripts that are predicted to act as competing-endogenous RNAs (ceRNAs) for tumor-suppressor genes. Our model-based analysis of the trans effect of 3′ UTR shortening (MAT3UTR) reveals a significant role in altering ceRNA expression. MAT3UTR predicts many trans-targets of 3′ UTR shortening, including PTEN, a crucial tumor-suppressor gene3 involved in ceRNA crosstalk4 with nine 3′UTR-shortening genes, including EPS15 and NFIA. Knockdown of NUDT21, a master 3′ UTR-shortening regulator2, represses tumor-suppressor genes such as PHF6 and LARP1 in trans in a miRNA-dependent manner. Together, the results of our analysis suggest a major role of 3′ UTR shortening in repressing tumor-suppressor genes in trans by disrupting ceRNA crosstalk, rather than inducing proto-oncogenes in cis.Shortening of mRNA 3′ UTRs is often observed in cancer. A combination of model-based analysis and experiments suggests that 3′ UTR shortening disrupts competing endogenous RNA crosstalk, thus influencing tumor-suppressor expression in trans.

Discovery and characterization of a novel CCND1/MRCK gene fusion in mantle cell lymphoma

The t(11;14) translocation resulting in constitutive cyclin D1 expression is an early event in mantle cell lymphoma (MCL) transformation. Patients with a highly proliferative phenotype produce cyclin D1 transcripts with truncated 3′UTRs that evade miRNA regulation. Here, we report the recurrence of a novel gene fusion in MCL cell lines and MCL patient isolates that consists of the full protein coding region of cyclin D1 (CCND1) and a 3′UTR consisting of sequences from both the CCND1 3′UTR and myotonic dystrophy kinase-related Cdc42-binding kinases (MRCK) intron one. The resulting CCND1/MRCK mRNA is resistant to CCND1-targeted miRNA regulation, and targeting the MRCK region of the chimeric 3′UTR with siRNA results in decreased CCND1 levels.

The drive to generate multiple forms of oncogenic cyclin D1 transcripts in mantle cell lymphoma

Alternative polyadenylation is a rapidly emerging form of gene regulation, which in its simplest form, enables the generation of mRNA transcripts that code for the same protein but have 3′UTRs of different lengths and regulatory content. For oncogenes, shorter 3′UTRs would be preferred as a mechanism to evade miRNA regulation. The shortening of the 3′UTR of cyclin D1 in mantle cell lymphoma offers provocative insights into this process. Patient samples have revealed that 3′UTR shortening may occur due to mutations, or translocations that result in the generation of a chimeric 3′UTR. The truncated cyclin D1 3′UTRs resulting from alternative polyadenylation, use a premature canonical polyadenylation signal close to the stop codon that was generated either as a result of mutations or provided by another gene in the chimeric 3′UTR. The sequence of the polyadenylation signal in mantle cell lymphoma appears to be critical for 3′end formation of the cyclin D1 transcript. Shortening the 3′UTR allows cyclin D1 to potentially evade regulation by over 80 miRNAs that are predicted to bind to its full length 3′UTR.

Abstract A13: Chemoprevention agent SHetA2 induces G1 arrest through modulation of a biological system driven by Cyclin D1

A13 Flexible heteroarotinoid (Flex-Het) compounds induce apoptosis through direct targeting of mitochondria and Bcl-2 proteins, inhibit angiogenesis through regulation of cytokine expression and induce differentiation through increased E-Cadherin expression. In addition, the lead Flex-Het, SHetA2, induces G1 cell cycle arrest through induction of Cyclin D1 protein phosphorylation, ubiquitination and degradation. Overexpression of Cyclin D1 abrogated the G1 arrest. SHetA2 counteraction of carcinogen-induced Cyclin D1 expression was associated with SHetA2 chemoprevention of transformation in an organotypic model. Cyclin D1 is a tightly controlled protein that drives G1 cell cycle progression. Overexpression of Cyclin D1 is frequently observed in ovarian cancer and is believed to be an early event in ovarian tumorigenesis. The hypothesis of this study was that SHetA2 induces G1 arrest through modulation of a biological system driven by Cyclin D1. Cyclin D1 degradation was associated with several expected down-stream events in 2 ovarian cancer cell lines. Co-IP assays demonstrated that Cyclin D1 loss was associated with relocation of p21 from the Cyclin D1 complex to the Cyclin E2 complex. Consistent with the stimulatory effect of p21 on the CyclinD1-Cdk4/6 complex and the inhibitory effect on the CyclinE2-Cdk2 kinase activity, Western blot analysis demonstrated that phosphorylation of Rb on serine 780, known to be induced by CyclinD1-Cdk4/6-p21, was decreased. This was followed by decreased Rb phosphorylation on serine 612, which is known to be induced by CyclinE2-Cdk2. SHetA2 inhibition of Cyclin A protein expression, confirmed that the repression of Rb phosphorylation prevented the release of E2F from Rb, thus preventing expression of S Phase genes. An rtPCR array demonstrated that 7 genes involved in cell cycle regulation were significantly altered above background in both ovarian cancer cell lines. Ingenuity analysis demonstrated that several of these genes are involved in an interacting biological system driving the cell cycle. A Cdk4 inhibitor, p16, and the down-stream gene Bax were up-regulated, while several Cyclin D1 and E2F interacting genes were also regulated. Study of upstream events driving Cyclin D1 degradation demonstrated that the GSK3β kinase, known to induce Cyclin D1 threonine 286 phosphorylation, was phosphorylated/inhibited by SHetA2 and not involved in the cell cycle arrest. This phosphorylation event could be due to the SHetA2 induced phosphorylation of Akt serine 473, which has been shown to lead to increased GSK3β phosphorylation. Experiments with libraries of chemical and siRNA kinase inhibitors demonstrated that inhibition of kinases, such as EGF-R, mTOR, PKC, DNA-PK, contributed to SHetA2 growth inhibition, depending on their expression and activity patterns in the specific cell lines evaluated. In conclusion, SHetA2 inhibition of Cyclin D1 induced a number of changes in down-stream and associated targets consistent with G1 arrest. Experiments are ongoing to identify the specific kinase responsible for SHetA2 induction of Cyclin D1 phosphorylation. Knowledge of SHetA2 modulation of the biological system regulating cell cycle progression provides important information, such as response biomarkers, that can be used in translational research associated with planned chemoprevention trials. Supported by CA106713 Citation Information: Cancer Prev Res 2008;1(7 Suppl):A13.