SV40 Small T Antigen Research Articles

Evaluating the Epithelial-Mesenchymal Program in Human Breast Epithelial Cells Cultured in Soft Agar Using a Novel Macromolecule Extraction Protocol.

Simple SummaryAnchorage-independent soft agar colony formation assays have been widely used as an in vitro surrogate for in vivo tumour formation in xenograft studies, and has found much utility in studies such as cancer drug development. However, molecular characterisation of cells grown in soft agar has proven difficult and sometimes even impossible. We developed a set of new methods that allow DNA, RNA and proteins (including phosphoproteins) to be extracted from cells grown in soft agar, even without visible colony formation. We used these methods to demonstrate the role of the epithelial-mesenchymal program in the malignant transformation of a classical human mammary epithelial cell model.The ability to grow in anchorage-independent conditions is an important feature of malignant cells, and it is well-established that cellular phenotypes in adherent cultures can differ widely from phenotypes observed in xenografts and anchorage-independent conditions. The anchorage-independent soft-agar colony formation assay has been widely used as a bridge between adherent cell cultures and animal tumor studies, providing a reliable in vitro tool to predict the tumorigenicity of cancer cells. However, this functional assay is limited in its utility for molecular mechanistic studies, as currently there is no reliable method that allows the extraction of biological macromolecules from cells embedded in soft-agar matrices, especially in experimental conditions where no visible colonies form. We developed a set of new methods that enable the extraction of DNA, RNA and proteins directly from cells embedded in soft agar, allowing for a wide range of molecular signaling analysis. Using the new methods and human mammary epithelial cells (HMECs), we studied the role of epithelial-mesenchymal transition (EMT) in the ability of HMECs to form colonies in soft agar. We found that, when cultured in soft agar instead of in adherent cultures, immortalized non-malignant HME-hTERT cells upregulated the epithelial program, which was noted to be necessary for their survival in this anchorage-independent condition. Overexpression of SV40 small T antigen (ST) or the EMT master-regulator SNAI1 negates this requirement and significantly enhances colony formation in soft agar driven by mutant-RAS. Interestingly, we found that, similar to SNAI1, ST also promotes EMT changes in HMECs, providing further support for EMT as a prerequisite for the efficient anchorage-independent colony formation driven by mutant-RAS in our HMEC model.

Immortalized normal human lung epithelial cell models for studying lung cancer biology.

Primary cultures of human lung epithelial cells are ideal representatives of normal lung epithelial cells, and while there are certain novel approaches for the long-term culture of lung epithelial cells, the cells eventually undergo irreversible growth arrest, limiting their experimental utility, particularly the ability to widely distribute these cultures and their clonal derivatives to the broader research community. Therefore, the establishment of immortalized normal human lung epithelial cell strains has garnered considerable attention. The number and type of oncogenic changes necessary for the tumorigenic transformation of normal cells could be determined using "normal" cell lines immortalized with the simian virus 40 (SV40) large T antigen (LT). A primary report suggested that LT, human telomerase reverse transcriptase (hTERT), and oncogenic RAS transformed normal lung epithelial cells into tumorigenic cells. Since LT inactivates the tumor suppressors p53 and RB, at least four alterations would be necessary. However, the SV40 small T antigen (ST), a different oncoprotein, was also introduced simultaneously with LT in the above-mentioned study. Furthermore, the possible uncharacterized functions of LT remained largely obscure. Therefore, no definitive conclusion could be arrived in these studies. Subsequent studies used methods that did not involve the use of oncoproteins and revealed that at least five genetic changes were necessary for full tumorigenic transformation. hTERT-immortalized normal human lung epithelial cell lines established without using viral oncoproteins were also used for investigating several aspects of lung cancer, such as epithelial to mesenchymal transition and the cancer stem cell theory. The use of immortalized normal lung epithelial cell models has improved our understanding of lung cancer pathogenesis and these models can serve as valuable research tools.

STRIPAK directs PP2A activity toward MAP4K4 to promote oncogenic transformation of human cells.

Alterations involving serine-threonine phosphatase PP2A subunits occur in a range of human cancers, and partial loss of PP2A function contributes to cell transformation. Displacement of regulatory B subunits by the SV40 Small T antigen (ST) or mutation/deletion of PP2A subunits alters the abundance and types of PP2A complexes in cells, leading to transformation. Here, we show that ST not only displaces common PP2A B subunits but also promotes A-C subunit interactions with alternative B subunits (B''', striatins) that are components of the Striatin-interacting phosphatase and kinase (STRIPAK) complex. We found that STRN4, a member of STRIPAK, is associated with ST and is required for ST-PP2A-induced cell transformation. ST recruitment of STRIPAK facilitates PP2A-mediated dephosphorylation of MAP4K4 and induces cell transformation through the activation of the Hippo pathway effector YAP1. These observations identify an unanticipated role of MAP4K4 in transformation and show that the STRIPAK complex regulates PP2A specificity and activity.

A fail-safe system to prevent oncogenesis by senescence is targeted by SV40 small T antigen.

Whereas large T antigen (LT) of simian virus 40 (SV40) promotes oncogenesis by inactivating the tumor suppressor proteins p53 and pRb, SV40 small T antigen (ST) has been thought to be dispensable for this process. However, here we show that LT promotes both oncogenic growth and senescence in human cells expressing oncogenic Ras and that this latter effect is antagonized by ST. Inactivation of p53 by LT alone promoted the senescence-associated secretory phenotype (SASP), whereas the additional expression of ST attenuated this phenotype, allowing cells to avoid oncogene-induced senescence (OIS) and thereby promoting efficient oncogenesis. ST interacts with and inhibits the function of heterochromatin protein 1-binding protein 3 (HP1BP3), a positive regulator of global microRNA biogenesis, and it thereby triggers aberrant upregulation of B-cell translocation gene 2 (BTG2), which is essential for prevention of SASP and OIS by ST. Collectively, our results indicate that the HP1BP3-BTG2 axis constitutes a fail-safe system to prevent oncogenesis by means of OIS induction, and that this system is hijacked by ST.

Abstract C132: Therapeutic reactivation of PP2A for prostate cancer treatment

Abstract Several new therapies have recently been approved for patients with castration-resistant prostate cancer (CRPC), however, none are curative and tumors ultimately develop resistance. Advances in the treatment of CRPC require novel approaches and therapies such as those outlined in this study. Most drug development efforts have focused on targeting single oncogenic proteins, an approach limited by the complexity of signaling networks and associated cross talk. Targeting phosphatases, the key negative regulators of signaling proteins, on the other hand, may overcome some of these limitations, particularly if these negative regulators themselves are altered.Through reverse engineering of tricyclic neuroleptic drugs, we have developed a series of small molecule activators of the serine/threonine phosphatase 2A (PP2A), a key negative regulator of numerous oncogenic signaling pathways. PP2A acts as a tumor suppressor and dephosphorylates several critical nodes in prostate cancer pathogenesis including the androgen receptor (AR). Decreased PP2A expression and/or activity have been correlated with castration-resistance in cell culture and human prostate cancer studies. These small molecule activators of PP2A (SMAPs), as represented by TRC-794, TRC-1154, and DT-061, directly bind and activate PP2A and have favorable pharmaceutical properties. In this study we sought to determine the activity of SMAPs in clinically relevant preclinical models of prostate cancer. Treatment of prostate cancer cell lines with SMAPs resulted in decreased cell viability and colony formation, cell cycle arrest, and an increase in apoptosis. Global Phosphoproteomic analysis of TRC-794 treated prostate cancer cells revealed that the AR and MYC were significantly perturbed in drug treated cells compared to controls which was subsequently confirmed by western blotting. Western blot analysis of prostate cancer cells demonstrated dose-dependent degradation of the AR resulting in PSA reduction and changes in canonical AR target gene expression. In order to investigate whether PP2A was mediating SMAP induced AR degradation, LNCAP cells were stably transduced with the SV40 small t antigen (ST), a potent oncoprotein that perturbs PP2A function. SMAPs were unable to degrade AR in LNCAP cells transduced with ST, suggesting that PP2A mediates SMAP induced AR degradation. SMAPs were evaluated in vivo in xenograft models representing prostate cancers that are sensitive to conventional therapy and resistant to enzalutamide, the current gold standard, due to overexpression of the AR or expression of androgen receptor splice variants (AR-SV). Single agent treatment with DT-1154 or DT-061 in vivo resulted in either significant tumor growth inhibition or tumor regression and induction of tumor cell apoptosis comparable to enzalutamide. Western blot analysis of the tumors demonstrated that the effects on tumor volume correlated strongly with target engagement as evidenced by significant decreases in PSA and AR expression in vivo. Additionally, these compounds demonstrated favorable pharmacokinetics and showed no overt toxicity. Combined these data highlight the potential for PP2A activation for both the treatment of CRPC and potentially for diverse PP2A inactivated tumor types and diseases. Citation Format: Kim McClinch, Rita Avelar, David Callejas, David Kastrinsky, Michael Ohlmeyer, Stephen Plymate, Matthew Galsky, Goutham Narla. Therapeutic reactivation of PP2A for prostate cancer treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C132.

P27kip1 Protein Levels Reflect a Nexus of Oncogenic Signaling during Cell Transformation

SV40 small t-antigen (ST) collaborates with SV40 large T-antigen (LT) and activated rasv12 to promote transformation in a variety of immortalized human cells. A number of oncogenes or the disruption of the general serine-threonine phosphatase protein phosphatase 2A (PP2A) can replace ST in this paradigm. However, the relationship between these oncogenes and PP2A activity is not clear. To address this, we queried the connectivity of these molecules in silico. We found that p27 was connected to each of those oncogenes that could substitute for ST. We further determined that p27 loss can substitute for the expression of ST during transformation of both rodent and human cells. Conversely, knock-in cells expressing the degradation-resistant S10A and T187A mutants of p27 were resistant to the transforming activities of ST. This suggests that p27 is an important target of the tumor-suppressive effects of PP2A and likely an important target of the multitude of cellular oncoproteins that emulate the transforming function of ST.

Comparisons between Murine Polyomavirus and Simian Virus 40 Show Significant Differences in Small T Antigen Function

Although members of a virus family produce similar gene products, those products may have quite different functions. Simian virus 40 (SV40) large T antigen (LT), for example, targets p53 directly, but murine polyomavirus LT does not. SV40 small T antigen (SVST) has received considerable attention because of its ability to contribute to transformation of human cells. Here, we show that there are major differences between SVST and polyomavirus small T antigen (POLST) in their effects on differentiation, transformation, and cell survival. Both SVST and POLST induce cell cycle progression. However, POLST also inhibits differentiation of 3T3-L1 preadipocytes and C2C12 myoblasts. Additionally, POLST induces apoptosis of mouse embryo fibroblasts. SVST reduces the proapoptotic transcriptional activity of FOXO1 through phosphorylation. On the other hand, SVST complements large T antigen and Ras for the transformation of human mammary epithelial cells (HMECs), but POLST does not. Mechanistically, the differences between SVST and POLST may lie in utilization of protein phosphatase 2A (PP2A). POLST binds both Aα and Aβ scaffolding subunits of PP2A while SVST binds only Aα. Knockdown of Aβ could mimic POLST-induced apoptosis. The two small T antigens can target different proteins for dephosphorylation. POLST binds and dephosphorylates substrates, such as lipins, that SVST does not.

Upregulation of miR-27a contributes to the malignant transformation of human bronchial epithelial cells induced by SV40 small T antigen

The introduction of the Simian virus 40 (SV40) early region, the telomerase catalytic subunit (hTERT) and an oncogenic allele of H-Ras directly transforms primary human cells. SV40 small T antigen (ST), which forms a complex with protein phosphatase 2A (PP2A) and inhibits PP2A activity, is believed to have a critical role in the malignant transformation of human cells. Recent evidence has shown that aberrant microRNA (miRNA) expression patterns are correlated with cancer development. Here, we identified miR-27a as a differentially expressed miRNA in SV40 ST-expressing cells. miR-27a is upregulated in SV40 ST-transformed human bronchial epithelial cells (HBERST). Suppression of miR-27a expression in HBERST cells or lung cancer cell lines (NCI-H226 and SK-MES-1) that exhibited high levels of miR-27a expression lead to cell growth arrested in the G(0)-G(1) phase. In addition, suppression of miR-27a in HBERST cells attenuated the capacity of such cells to grow in an anchorage-independent manner. We also found that suppression of the PP2A B56γ expression resulted in upregulation of miR-27a similar to that achieved by the introduction of ST, indicating that dysregulation of miR-27a expression in ST-expressing cells was mediated by the ST-PP2A interaction. Moreover, we discovered that Fbxw7 gene encoding F-box/WD repeat-containing protein 7 was a potential miR-27a target validated by dual-luciferase reporter system analysis. The inverse correlation between miR-27a expression levels and Fbxw7 protein expression was further confirmed in both cell models and human tumor samples. Fbxw7 regulates cell-cycle progression through the ubiquitin-dependent proteolysis of a set of substrates, including c-Myc, c-Jun, cyclin E1 and Notch 1. Thus, promotion of cell growth arising from the suppression of Fbxw7 by miR-27a overexpression might be responsible for the viral oncoprotein ST-induced malignant transformation. These observations demonstrate that miR-27a functions as an oncogene in human tumorigenesis.

CPLA2 Regulates the Expression of Type I Interferons and Intracellular Immunity to Chlamydia trachomatis

Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase RAS and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA(2) are activated and are important for the onset of inflammatory responses. In this study we tested if activation of ERK and cPLA(2) occurred as a result of RAS signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. We provide genetic and pharmacological evidence that during infection RAS, ERK, and, to a lesser extent, cPLA(2) activation are uncoupled, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA(2) signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells, inhibition of ERK and cPLA(2) played a significant protective role against C. trachomatis. We determined that cPLA(2)-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of beta interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Furthermore, the MAPK p38 was primarily responsible for cPLA(2) activation in Chlamydia-infected cells and IRG expression. Overall, these findings define a previously unrecognized role for cPLA(2) in the induction of cell autonomous cellular immunity to Chlamydia and highlight the many non-canonical signaling pathways engaged during infection.

Clonal Selection in Malignant Transformation of Human Fibroblasts Transduced with Defined Cellular Oncogenes

Recent evidence has implied that disruption of a limited number of defined cellular pathways is necessary and sufficient for neoplastic conversion of a variety of normal human cell types in tissue culture. We show instead that malignancy in such models results from an iterative process of clonal selection in vitro and/or in vivo. Normal human fibroblasts underwent malignant transformation after transduction with telomerase, cyclin-dependent kinase 4, dominant-negative p53, and activated Ras or MEK. Furthermore, culture conditions favoring overgrowth resulted in clonal selection, which with added Ras or MEK oncogenes led to the emergence of tumorigenic clones. Such tumors showed variable degrees of malignancy with some even exhibiting metastasis. SV40 small t antigen (ST) has been reported to be necessary and sufficient to convert human fibroblasts with these pathway aberrations to a polyclonal tumor. However, we observed that clonal tumors emerged even with ST addition. Genomic instability was markedly increased by p53 and Rb pathway abrogation. Under the same conditions, fibroblasts with these alterations failed to induce tumors, implying that genomic instability may be necessary but not sufficient for malignant transformation. These findings indicate that the minimum number of events required for malignant transformation of human fibroblasts is greater than has been enumerated by such oncogene addition strategies and support a stochastic cancer progression model initiated by four defined cellular alterations.

Structural Basis of PP2A Inhibition by Small t Antigen

The SV40 small t antigen (ST) is a potent oncoprotein that perturbs the function of protein phosphatase 2A (PP2A). ST directly interacts with the PP2A scaffolding A subunit and alters PP2A activity by displacing regulatory B subunits from the A subunit. We have determined the crystal structure of full-length ST in complex with PP2A A subunit at 3.1 Å resolution. ST consists of an N-terminal J domain and a C-terminal unique domain that contains two zinc-binding motifs. Both the J domain and second zinc-binding motif interact with the intra-HEAT-repeat loops of HEAT repeats 3–7 of the A subunit, which overlaps with the binding site of the PP2A B56 subunit. Intriguingly, the first zinc-binding motif is in a position that may allow it to directly interact with and inhibit the phosphatase activity of the PP2A catalytic C subunit. These observations provide a structural basis for understanding the oncogenic functions of ST.

Structural and biochemical insights into the regulation of protein phosphatase 2A by small t antigen of SV40

The small t antigen (ST) of DNA tumor virus SV40 facilitates cellular transformation by disrupting the functions of protein phosphatase 2A (PP2A) through a poorly defined mechanism. The crystal structure of the core domain of SV40 ST bound to the scaffolding subunit of human PP2A reveals that the ST core domain has a novel zinc-binding fold and interacts with the conserved ridge of HEAT repeats 3-6, which overlaps with the binding site for the B' (also called PR61 or B56) regulatory subunit. ST has a lower binding affinity than B' for the PP2A core enzyme. Consequently, ST does not efficiently displace B' from PP2A holoenzymes in vitro. Notably, ST inhibits PP2A phosphatase activity through its N-terminal J domain. These findings suggest that ST may function mainly by inhibiting the phosphatase activity of the PP2A core enzyme, and to a lesser extent by modulating assembly of the PP2A holoenzymes.

Polyoma and SV40 proteins differentially regulate PP2A to activate distinct cellular signaling pathways involved in growth control

Binding of Src family kinases to membrane-associated polyoma virus middle T-antigen (PyMT) can result in the phosphorylation of PyMT tyrosine 250, which serves as a docking site for the binding of Shc and subsequent activation of the Raf-MEK-ERK (MAP) kinase cascade. In a screen for PyMT variants that could not activate the ARF tumor suppressor, we isolated a cytoplasmic nontransforming mutant (MTA) that encoded a C-terminal truncated form of the PyMT protein. Surprisingly, MTA was able to strongly activate the MAP kinase pathway in the absence of Src family kinase and Shc binding. Interestingly, the polyoma small T-antigen (PyST), which shares with MTA both partial amino acid sequence homology and cellular location, also activates the MAP kinase cascade. Activation of the MAP kinase cascade by both MTA and PyST has been demonstrated to be PP2A-dependent. Neither MTA nor PyST activate the phosphorylation of AKT. The SV40 small T-antigen, which is similar to PyST in containing a J domain and in binding to the PP2A AC dimer, does not activate the MAP kinase cascade, but does stimulate phosphorylation of AKT in a PP2A-dependent manner. These findings highlight a novel role of PP2A in stimulating the MAP kinase cascade and indicate that the similar polyoma and SV40 small T-antigens influence PP2A to activate discrete cellular signaling pathways involved in growth control.

Inhibition of PP2A, but not PP5, mediates p53 activation by low levels of okadaic acid in rat liver epithelial cells

The microbial toxin okadaic acid (OA) specifically inhibits PPP-type ser/thr protein phosphatases. OA is an established tumor promoter with numerous cellular effects that include p53-mediated cell cycle arrest. In T51B rat liver epithelial cells, a model useful for tumor promotion studies, p53 activation is induced by tumor-promoting (low nanomolar) concentrations of OA. Two phosphatases sensitive to these concentrations of OA, PP2A and protein phosphatase 5 (PP5), have been implicated as negative regulators of p53. In this study we examined the respective roles of these phosphatases in p53 activation in non-neoplastic T51B cells. Increases in p53 activity were deduced from levels of p21 (cip1) and/or the rat orthologue of mdm2, two p53-regulated gene products whose induction was blocked by siRNA-mediated knockdown of p53. As observed with 10 nM OA, both phospho-ser15-p53 levels and p53 activity were increased by 10 microM fostriecin or SV40 small t-antigen. Both of these treatments selectively inhibit PP2A but not PP5. siRNA-mediated knockdown of PP2A, but not PP5, also increased p53 activity. Finally, adenoviral-mediated over-expression of an OA-resistant form of PP5 did not prevent increased phospho-ser15-p53, p53 protein, or p53 activity caused by 10 nM OA. Together these results indicate that PP5 blockade is not responsible for OA-induced p53 activation and G1 arrest in T51B cells. In contrast, specific blockade of PP2A mimics p53-related responses to OA in T51B cells, suggesting that PP2A is the target for this response to OA.

MTOR-dependent Suppression of Protein Phosphatase 2A Is Critical for Phospholipase D Survival Signals in Human Breast Cancer Cells

A critical aspect of tumor progression is the generation of survival signals that overcome default apoptotic programs. Recent studies have revealed that elevated phospholipase D activity generates survival signals in breast and perhaps other human cancers. We report here that the elevated phospholipase D activity in the human breast cancer cell line MDA-MB-231 suppresses the activity of the putative tumor suppressor protein phosphatase 2A in a mammalian target of rapamycin (mTOR)-dependent manner. Increasing the phospholipase D activity in MCF7 cells also suppressed protein phosphatase 2A activity. Elevated phospholipase D activity suppressed association of protein phosphatase 2A with both ribosomal subunit S6-kinase and eukaryotic initiation factor 4E-binding protein 1. Suppression of protein phosphatase 2A by SV40 small t-antigen has been reported to be critical for the transformation of human cells with SV40 early region genes. Consistent with a critical role for protein phosphatase 2A in phospholipase D survival signals, either SV40 small t-antigen or pharmacological suppression of protein phosphatase 2A restored survival signals lost by the suppression of either phospholipase D or mTOR. Blocking phospholipase D signals also led to reduced phosphorylation of the pro-apoptotic protein BAD at the protein phosphatase 2A dephosphorylation site at Ser-112. The ability of phospholipase D to suppress protein phosphatase 2A identifies a critical target of an emerging phospholipase D/mTOR survival pathway in the transformation of human cells.

Tumorigenic study on hepatocytes coexpressing SV40 with Ras

A model of neoplastic transformation by the combination of SV40 large T antigen (LT), SV40 small T antigen (ST), oncogenic Ras, and human telomerase reverse trasncriptase subunit (hTERT) has become established and replicated in primary human fibroblasts, however, there is no report on human hepatocytes. Here we use cell transplantation model, and show that transplantation of human hepatocytes of HL-7702 and HL-7703 expressing Ha-RasV12 and SV40 LT into subrenal capsule of immunodeficient mice results in fully malignant tumors, in contrast to conventional subcutaneous injections where tumors fail to develop. In GM-847 cell study, we have found that hTERT is not required for tumorigenic growth in subrenal capsule transplantation, however, it is required in subcutaneous injection assay. These results demonstrate that Human hepatocytes can be transformed under kidney capsule by coexpressing SV40 LT and Ha-RasV12, neither hTERT nor protein phosphatase 2A (PP2A) inhibition are required for malignant transformation, a gene which increases cell survival in the subcutaneous injection model is not required for tumorigenic growth in subrenal capsule.

Activation of the Canonical Wnt Pathway during Genital Keratinocyte Transformation: A Model for Cervical Cancer Progression

Cervical carcinoma, the second leading cause of cancer deaths in women worldwide, is associated with human papillomavirus (HPV). HPV-infected individuals are at high risk for developing cervical carcinoma; however, the molecular mechanisms that lead to the progression of cervical cancer have not been established. We hypothesized that in a multistep carcinogenesis model, HPV provides the initial hit and activation of canonical Wnt pathway may serve as the second hit. To test this hypothesis, we evaluated the canonical Wnt pathway as a promoting factor of HPV-induced human keratinocyte transformation. In this in vitro experimental cervical carcinoma model, primary human keratinocytes immortalized by HPV were transformed by SV40 small-t (smt) antigen. We show that smt-transformed cells have high cytoplasmic beta-catenin levels, a hallmark of activated canonical Wnt pathway, and that activation of this pathway by smt is mediated through its interaction with protein phosphatase-2A. Furthermore, inhibition of downstream signaling from beta-catenin inhibited the smt-induced transformed phenotype. Wnt pathway activation transformed HPV-immortalized primary human keratinocytes even in the absence of smt. However, activation of the Wnt pathway in the absence of HPV was not sufficient to induce transformation. We also detected increased cytoplasmic and nuclear staining of beta-catenin in invasive cervical carcinoma samples from 48 patients. We detected weak cytoplasmic and no nuclear staining of beta-catenin in 18 cases of cervical dysplasia. Our results suggest that the transformation of HPV expressing human keratinocytes requires activation of the Wnt pathway and that this activation may serve as a screening tool in HPV-positive populations to detect malignant progression.

PP2A-dependent transactivation of the cyclin A promoter by SV40 ST is mediated by a cell cycle-regulated E2F site

The Simian Virus 40 (SV40) small-t antigen (ST) plays an important role in driving cell proliferation, enhancing transformation by the large-T (LT) antigen. Potential targets of ST are the cyclin kinase inhibitor p27 and the cyclin A gene itself. Transactivation of the cyclin A promoter by ST depends on the interaction of ST with protein phosphatase 2A (PP2A) and occurs through a cell cycle-regulated E2F site near the transcription start site of the promoter. A third SV40 early protein, 17KT, also transactivates the cyclin A promoter but, in this case, transactivation depends on the dnaJ domain of the protein.

Identification of specific PP2A complexes involved in human cell transformation

The SV40 small t antigen (ST) interacts with the serine-threonine protein phosphatase 2A (PP2A). To investigate the role of this interaction in transformation, we suppressed the expression of the PP2A B56γ subunit in human embryonic kidney (HEK) epithelial cells expressing SV40 large T antigen, hTERT, and H-RAS. Suppression of PP2A B56γ expression inhibited PP2A-specific phosphatase activity similar to that achieved by ST and conferred the ability to grow in an anchorage-independent fashion and to form tumors. Overexpression of PP2A B56γ3 in tumorigenic HEK cells expressing ST or human lung cancer cell lines partially reversed the tumorigenicity of these cells. These observations identify specific PP2A complexes involved in human cell transformation.

Enumeration of the simian virus 40 early region elements necessary for human cell transformation.

While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.