Ole Gjoerup

T antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus

Merkel cell polyomavirus (MCV) is a virus discovered in our laboratory at the University of Pittsburgh that is monoclonally integrated into the genome of ≈80% of human Merkel cell carcinomas (MCCs). Transcript mapping was performed to show that MCV expresses transcripts in MCCs similar to large T (LT), small T (ST), and 17kT transcripts of SV40. Nine MCC tumor-derived LT genomic sequences have been examined, and all were found to harbor mutations prematurely truncating the MCV LT helicase. In contrast, four presumed episomal viruses from nontumor sources did not possess this T antigen signature mutation. Using coimmunoprecipitation and origin replication assays, we show that tumor-derived virus mutations do not affect retinoblastoma tumor suppressor protein (Rb) binding by LT but do eliminate viral DNA replication capacity. Identification of an MCC cell line (MKL-1) having monoclonal MCV integration and the signature LT mutation allowed us to functionally test both tumor-derived and wild type (WT) T antigens. Only WT LT expression activates replication of integrated MCV DNA in MKL-1 cells. Our findings suggest that MCV-positive MCC tumor cells undergo selection for LT mutations to prevent autoactivation of integrated virus replication that would be detrimental to cell survival. Because these mutations render the virus replication-incompetent, MCV is not a “passenger virus” that secondarily infects MCC tumors.

Human Merkel cell polyomavirus infection I. MCV T antigen expression in Merkel cell carcinoma, lymphoid tissues and lymphoid tumors

Merkel cell polyomavirus (MCV) is a recently discovered human virus closely related to African green monkey lymphotropic polyomavirus. MCV DNA is integrated in ∼80% of Merkel cell carcinomas (MCC), a neuroendocrine skin cancer linked to lymphoid malignancies such as chronic lymphocytic leukemia (CLL). To assess MCV infection and its association with human diseases, we developed a monoclonal antibody that specifically recognizes endogenous and transfected MCV large T (LT) antigen. We show expression of MCV LT protein localized to nuclei of tumor cells from MCC having PCR quantified MCV genome at an average of 5.2 (range 0.8–14.3) T antigen DNA copies per cell. Expression of this putative viral oncoprotein in tumor cells provides the mechanistic underpinning supporting the notion that MCV causes a subset of MCC. In contrast, although 2.2% of 325 hematolymphoid malignancies surveyed also showed evidence for MCV infection by DNA PCR, none were positive at high viral copy numbers, and none of 173 lymphoid malignancies examined on tissue microarrays expressed MCV LT protein in tumor cells. As with some of the other human polyomaviruses, lymphocytes may serve as a tissue reservoir for MCV infection, but hematolymphoid malignancies associated with MCC are unlikely to be caused by MCV.

Human mammary epithelial cell transformation through the activation of phosphatidylinositol 3-kinase

Recent studies have demonstrated that introduction of hTERT in combination with SV40 large T antigen (LT), small t antigen (st), and H-rasV12 suffices to transform many primary human cells. In human mammary epithelial cells (HMECs) expressing elevated c-Myc, activated H-Ras is dispensable for anchorage-independent growth. Using this system, we show that st activates the PI3K pathway and that constitutive PI3K signaling substitutes for st in transformation. Moreover, using constitutively active versions of Akt1 and Rac1, we show that these downstream pathways of PI3K synergize to achieve anchorage-independent growth. At lower levels of c-myc expression, activated PI3K also replaces st to complement H-rasV12 and LT and confers both soft agar growth and tumorigenicity. However, elevated c-myc expression cannot replace H-rasV12 for tumorigenesis. These observations begin to define the pathways perturbed during the transformation of HMECs.

The p110α isoform of PI3K is essential for proper growth factor signaling and oncogenic transformation

Growth factor signaling is mediated through Class IA phosphatidylinositol 3-kinases (PI3Ks). Among this class of enzymes, only p110α, encoded by the PIK3CA gene, has been found to be mutant in human cancers. To determine the specific functions of p110α, we generated mice carrying a conditionally targeted allele of the PIK3CA gene. Here, we report that PIK3CA-knockout mouse embryonic fibroblasts are deficient in cellular signaling in response to various growth factors, unable to differentiate into adipocytes, and resistant to oncogenic transformation induced by a variety of oncogenic receptor tyrosine kinases, indicating a fundamental role for p110α in these biological processes.

Update on human polyomaviruses and cancer.

Over 50 years of polyomavirus research has produced a wealth of insights into not only general biologic processes in mammalian cells, but also, how conditions can be altered and signaling systems tweaked to produce transformation phenotypes. In the past few years three new members (KIV, WUV, and MCV) have joined two previously known (JCV and BKV) human polyomaviruses. In this review, we present updated information on general virologic features of these polyomaviruses in their natural host, concentrating on the association of MCV with human Merkel cell carcinoma. We further present a discussion on advances made in SV40 as the prototypic model, which has and will continue to inform our understanding about viruses and cancer.

SIK1 Couples LKB1 to p53-Dependent Anoikis and Suppresses Metastasis

Loss of SIK1 suppresses cell death resulting from loss of cell anchorage and promotes metastasis in the absence of primary tumors. Preventing Metastatic Spread The tumor suppressor p53 promotes anoikis, the apoptotic death of cells that lose adhesion and become detached from their appropriate location, thereby suppressing cancer metastasis. Here, Cheng et al. show that the serine-threonine kinase SIK1 (salt-inducible kinase 1) regulates p53-dependent anoikis. Loss of SIK1 facilitated the anchorage-independent growth of cultured cells and promoted the development of micrometastases in the absence of primary tumors in immunodeficient mice. Moreover, decreased SIK1 expression correlated with appearance of distal metastases in human breast cancer. The authors propose that SIK1, by promoting anoikis, prevents the dissemination of precancerous cells and thereby plays a critical role in inhibiting metastasis. Resistance to anoikis, the subtype of apoptosis triggered by lack of adhesion, contributes to malignant transformation and the development of metastasis. Although several lines of evidence suggest that p53 plays a critical role in anoikis, the pathway(s) that connect cell detachment to p53 remain undefined. Here, through the use of a kinome-wide loss-of-function screen, we identify the serine-threonine kinase SIK1 (salt-inducible kinase 1) as a regulator of p53-dependent anoikis. Inactivation of SIK1 compromised p53 function in anoikis and allowed cells to grow in an anchorage-independent manner. In vivo, SIK1 loss facilitated metastatic spread and survival of disseminated cells as micrometastases in lungs. The presence of functional SIK1 was required for the activity of the kinase LKB1 in promoting p53-dependent anoikis and suppressing anchorage-independent growth, Matrigel invasion, and metastatic potential. In human cancers, decreased expression of the gene encoding SIK1 closely correlated with development of distal metastases in breast cancers from three independent cohorts. Together, these findings indicate that SIK1 links LKB1 to p53-dependent anoikis and suppresses metastasis.

Simian Virus 40 Large T Antigen Disrupts Genome Integrity and Activates a DNA Damage Response via Bub1 Binding

ABSTRACT Simian virus 40 (SV40) large T antigen (LT) is a multifunctional protein that is important for viral replication and oncogenic transformation. Previously, infection of monkey or human cells with SV40 was shown to lead to the induction of DNA damage response signaling, which is required for efficient viral replication. However, it was not clear if LT is sufficient to induce the damage response and, if so, what the genetic requirements and functional consequences might be. Here, we show that the expression of LT alone, without a replication origin, can induce key DNA damage response markers including the accumulation of γ-H2AX and 53BP1 in nuclear foci. Other DNA damage-signaling components downstream of ATM/ATR kinases were induced, including chk1 and chk2. LT also bound the Claspin mediator protein, which normally facilitates the ATR activation of chk1 and monitors cellular replication origins. Stimulation of the damage response by LT depends mainly on binding to Bub1 rather than to the retinoblastoma protein. LT has long been known to stabilize p53 despite functionally inactivating it. We show that the activation of a DNA damage response by LT via Bub1 appears to play a major role in p53 stabilization by promoting the phosphorylation of p53 at Ser15. Accompanying the DNA damage response, LT induces tetraploidy, which is also dependent on Bub1 binding. Taken together, our data suggest that LT, via Bub1 binding, breaches genome integrity mechanisms, leading to DNA damage responses, p53 stabilization, and tetraploidy.

Protein phosphatase 2A regulates life and death decisions via Akt in a context-dependent manner

Here, we show how targeting protein phosphatase 2A (PP2A), a key regulator of cellular protein phosphorylation, can either induce or prevent apoptosis depending on what other signals the cell is receiving. The oncoprotein polyoma small T interacts with PP2A to regulate survival. In the presence of growth factors, small T induces apoptosis. Akt activity, which usually promotes survival, is required for this death response, because inhibitors of Akt or PI3 kinase protect cells from death. The activation of Akt under these conditions is partial, characterized by T308 phosphorylation but not S473 phosphorylation. In the absence of growth factors, small T protects from cell death. Here, small T uses PP2A to promote phosphorylation of Akt on both T308 and S473. This effect results in a different pattern of phosphorylation of Akt substrates and shifts Akt from a proapoptotic (presence of growth factors) to an antiapoptotic mode (absence of growth factors). An intriguing possibility is that Akt phosphorylation could be therapeutically disregulated to decrease the survival of cancer cells.

Rac and Cdc42 Are Potent Stimulators of E2F-dependent Transcription Capable of Promoting Retinoblastoma Susceptibility Gene Product Hyperphosphorylation

The Rho family of GTPases plays an important and diverse role in reorganization of the actin cytoskeleton, transcriptional regulation, and multiple aspects of cell growth. Our study has examined their potential links to the cell cycle machinery. We find that constitutively active mutants of Rac and Cdc42, but not Rho, are potent inducers of E2F transcriptional activity in NIH 3T3 fibroblasts. Furthermore, activated Rac and Cdc42, but again not Rho, are capable of inducing cyclin D1 accumulation and pRB hyperphosphorylation in serum-deprived cells, outlining one route leading to enhanced E2F-mediated transcription. The inhibitory effect of the cyclin-dependent kinase inhibitors, p16 ink4 , p21 cip1 , and p27 cip on Rac/Cdc42-mediated E2F transcription corroborates a role for pRB family members and their functional inactivation by cyclin-dependent kinases in generating E2F activity. While the up-regulation of E2F transcriptional activity by Rac or Cdc42, not Rho, suffices for entry into S phase and DNA synthesis in Rat-1 R12 cells, this is clearly not the case in NIH 3T3, where additional requirements must exist.

Cellular and Viral Factors Regulating Merkel Cell Polyomavirus Replication

Merkel cell polyomavirus (MCV), a previously unrecognized component of the human viral skin flora, was discovered as a mutated and clonally-integrated virus inserted into Merkel cell carcinoma (MCC) genomes. We reconstructed a replicating MCV clone (MCV-HF), and then mutated viral sites required for replication or interaction with cellular proteins to examine replication efficiency and viral gene expression. Three days after MCV-HF transfection into 293 cells, although replication is not robust, encapsidated viral DNA and protein can be readily isolated by density gradient centrifugation and typical ∼40 nm diameter polyomavirus virions are identified by electron microscopy. The virus has an orderly gene expression cascade during replication in which large T (LT) and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. VP1 and sT proteins are not detected, and spliced 57kT is markedly diminished, in the replication-defective virus suggesting that early gene splicing and late gene transcription may be dependent on viral DNA replication. MCV replication and encapsidation is increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. Mutation of the MCV LT vacuolar sorting protein hVam6p (Vps39) binding site also enhances MCV replication while exogenous hVam6p overexpression reduces MCV virion production by >90%. Although MCV-HF generates encapsidated wild-type MCV virions, we did not find conditions for persistent transmission to recipient cell lines suggesting that MCV has a highly restricted tropism. These studies identify and highlight the role of polyomavirus DNA replication in viral gene expression and show that viral sT and cellular hVam6p are important factors regulating MCV replication. MCV-HF is a molecular clone that can be readily manipulated to investigate factors affecting MCV replication.