Merkel cell polyomavirus (MCV) is a virus discovered in our laboMerkel cell carcinoma ͉ pRB interaction ͉ viral integration ͉ virus replication ͉ helicase M erkel cell carcinoma (MCC) is an aggressive skin cancer associated with sun exposure and immunosuppression (1, 2). Using digital transcriptome subtraction, we recently identified Merkel cell polyomavirus (MCV) as a novel polyomavirus integrated into the genome of MCC tumors (3, 4). The close association between MCV and MCC has been confirmed by others (5). Polyomaviruses are small circular DNA viruses encoding a T antigen oncoprotein locus. T antigens are expressed from variably spliced viral transcripts that target tumor suppressor and cell cycle regulatory proteins, including retinoblastoma tumor suppressor protein (Rb) (6), p53 (7, 8), protein phosphatase 2A (9), and Bub1 (10). Murine polyomavirus (MuPyV) middle T (MT) antigen, a membrane-bound protein, is particularly potent in initiating cell transformation through interactions with phosphatidylinositol 3-kinase, protein phosphatase 2A, Src, and Shc proteins (11,12). MCV large T (LT) antigen retains conserved domains, such as pocket Rb binding LXCXE and DnaJ motifs, present across virus species (13). LT not only encodes tumor suppressor targeting domains but also origin binding and helicase/ATPase functions required for viral genome replication. MCV integration in MCC tumors is incompatible with transmissible virus and likely represents a rare biological accident in which the tumor cell is a dead-end host. The monoclonal pattern of MCV integration into MCC tumors suggests that virus integration occurs before tumor cell expansion and that MCV is a contributing factor in a portion of MCC (3).We have isolated MCV T antigen sequences from both tumor cases and nontumor cases to characterize their abilities to act as a viral DNA replicase. Sequence analysis demonstrates that LT protein is prematurely truncated in all MCC cases, whereas the Rb-interacting domain is preserved. Viruses from nontumor sources do not possess these mutations. We also describe here an MCC cell line stably harboring MCV and an origin replication assay to assess MCV replication. We show that wild type (WT) LT from nontumorous sources activates MCV replication of integrated tumor virus, suggesting that MCV-associated MCC arises from a two-step process in which viral genome integrates into the host genome and develops T antigen mutations to prevent autonomous viral genome replication. Failure to truncate the viral T antigen may lead to DNA damage responses or immune recognition that hinders nascent tumor cell survival.

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. ' 2009 UICC

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.

Growth factor signaling is mediated through Class IA phosphatidylinositol 3-kinases (PI3Ks). Among this class of enzymes, only p110␣, encoded by the PIK3CA gene, has been found to be mutant in human cancers. To determine the specific functions of p110␣, 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 p110␣ in these biological processes.adipocyte differentiation ͉ cancer therapy ͉ conditional knockout ͉ PIK3CA C lass IA PI3Ks are heterodimeric lipid kinases consisting of a p110 catalytic subunit complexed to one of a family of regulatory subunits (p85␣, p55␣, p50␣, p85␤, p55␥), collectively called p85 (1, 2). In response to growth factor stimulation and the subsequent activation of receptor tyrosine kinases (RTKs), class IA PI3Ks are recruited to the membrane via interaction of the p85 subunit with phosphotyrosine-containing motifs on the activated receptor. The p110 catalytic subunit of PI3K then catalyzes the phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to form phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). The lipid second messenger PIP 3 in turn activates the Ser͞Thr kinase AKT and other downstream effectors to regulate multiple cellular functions, including proliferation, survival and migration. There are three isoforms of the p110 catalytic subunit (␣, ␤, and ␦) that are encoded by the PIK3CA, PIK3CB, and PIK3CD genes, respectively. Both p110␣ and p110␤ isoforms are ubiquitously expressed, whereas the p110␦ subunit is primarily found in leukocytes (3, 4). Notably, among these isoforms, only p110␣ has been found to be frequently mutated in human tumors (5), suggesting distinct roles for the individual p110 isoforms in both normal signaling and oncogenic transformation. Previous efforts using gene targeting in mice have revealed that p110␦ plays essential roles in the development and function of lymphocytes, mast cells and possibly neutrophils (6-10). However, mice lacking p110␣ or p110␤ die early in embryonic development (11,12), which has precluded delineating the functions of the individual p110␣ and p110␤ isoforms by genetic ablation. Recent articles have used either a mouse heterozygous for the knockin of a kinase dead allele of p110␣ (13) or small molecule inhibitors of PI3K-p110␣ (14) to study its role in insulin signaling. Here, we report, for the first time, the effects of complete genetic ablation of PI3K-p110␣ on signaling elicited by a panel of growth factors, adipocyte differentiation and oncogenic transformation. Strikingly, we observe that knockout of this single isoform is capable of blocking both normal and oncogenic growth factor signal pathways. Results and DiscussionTo investigate the specific role of p110␣ in signaling an...