Antonio Di Cristofano

Pten is essential for embryonic development and tumour suppression.

The PTEN gene encodes a dual-specificity phosphatase mutated in a variety of human cancers. PTEN germline mutations are found in three related human autosomal dominant disorders, Cowden disease (CD), Lhermitte-Duclos disease (LDD) and Bannayan-Zonana syndrome (BZS), characterized by tumour susceptibility and developmental defects. To examine the role of PTEN in ontogenesis and tumour suppression, we disrupted mouse Pten by homologous recombination. Pten inactivation resulted in early embryonic lethality. Pten–/– ES cells formed aberrant embryoid bodies and displayed an altered ability to differentiate into endodermal, ectodermal and mesodermal derivatives. Pten+/– mice and chimaeric mice derived from Pten+/– ES cells showed hyperplastic-dysplastic changes in the prostate, skin and colon, which are characteristic of CD, LDD and BZS. They also spontaneously developed germ cell, gonadostromal, thyroid and colon tumours. In addition, Pten inactivation enhanced the ability of ES cells to generate tumours in nude and syngeneic mice, due to increased anchorage-independent growth and aberrant differentiation. These results support the notion that PTEN haploinsufficiency plays a causal role in CD, LDD and BZS pathogenesis, and demonstrate that Pten is a tumour suppressor essential for embryonic development.

Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association.

The PTEN tumor suppressor is mutated in diverse human cancers and in hereditary cancer predisposition syndromes. PTEN is a phosphatase that can act on both polypeptide and phosphoinositide substrates in vitro. The PTEN structure reveals a phosphatase domain that is similar to protein phosphatases but has an enlarged active site important for the accommodation of the phosphoinositide substrate. The structure also reveals that PTEN has a C2 domain. The PTEN C2 domain binds phospholipid membranes in vitro, and mutation of basic residues that could mediate this reduces PTENs membrane affinity and its ability to suppress the growth of glioblastoma tumor cells. The phosphatase and C2 domains associate across an extensive interface, suggesting that the C2 domain may serve to productively position the catalytic domain on the membrane.

Activation of AKT Kinases in Cancer: Implications for Therapeutic Targeting

The AKT1, AKT2, and AKT3 kinases have emerged as critical mediators of signal transduction pathways downstream of activated tyrosine kinases and phosphatidylinositol 3-kinase. An ever-increasing list of AKT substrates has precisely defined the multiple functions of this kinase family in normal physiology and disease states. Cellular processes regulated by AKT include cell proliferation and survival, cell size and response to nutrient availability, intermediary metabolism, angiogenesis, and tissue invasion. All these processes represent hallmarks of cancer, and a burgeoning literature has defined the importance of AKT alterations in human cancer and experimental models of tumorigenesis, continuing the legacy represented by the original identification of v-Akt as the transforming oncogene of a murine retrovirus. Many oncoproteins and tumor suppressors intersect in the AKT pathway, finely regulating cellular functions at the interface of signal transduction and classical metabolic regulation. This careful balance is altered in human cancer by a variety of activating and inactivating mechanisms that target both AKT and interrelated proteins. Reprogramming of this altered circuitry by pharmacologic modulation of the AKT pathway represents a powerful strategy for rational cancer therapy. In this review, we summarize a large body of data, from many types of cancer, indicating that AKT activation is one of the most common molecular alterations in human malignancy. We also review mechanisms of activation of AKT kinases, examples of therapeutic modulation of the AKT pathway in animal models, and the current status of efforts to target molecular components of the AKT pathway for cancer therapy and, possibly, cancer prevention.

Pten Dose Dictates Cancer Progression in the Prostate

Complete inactivation of the PTEN tumor suppressor gene is extremely common in advanced cancer, including prostate cancer (CaP). However, one PTEN allele is already lost in the vast majority of CaPs at presentation. To determine the consequence of PTEN dose variations on cancer progression, we have generated by homologous recombination a hypomorphic Pten mouse mutant series with decreasing Pten activity: Ptenhy/+ > Pten+/− > Ptenhy/− (mutants in which we have rescued the embryonic lethality due to complete Pten inactivation) > Pten prostate conditional knockout (Ptenpc) mutants. In addition, we have generated and comparatively analyzed two distinct Ptenpc mutants in which Pten is inactivated focally or throughout the entire prostatic epithelium. We find that the extent of Pten inactivation dictate in an exquisite dose-dependent fashion CaP progression, its incidence, latency, and biology. The dose of Pten affects key downstream targets such as Akt, p27Kip1, mTOR, and FOXO3. Our results provide conclusive genetic support for the notion that PTEN is haploinsufficient in tumor suppression and that its dose is a key determinant in cancer progression.

Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse

The genetic bases underlying prostate tumorigenesis are poorly understood. Inactivation of the tumor-suppressor gene PTEN and lack of p27KIP1 expression have been detected in most advanced prostate cancers. But mice deficient for Cdkn1b (encoding p27Kip1) do not develop prostate cancer. PTEN activity leads to the induction of p27KIP1 expression, which in turn can negatively regulate the transition through the cell cycle. Thus, the inactivation of p27KIP1 may be epistatic to PTEN in the control of the cell cycle. Here we show that the concomitant inactivation of one Pten allele and one or both Cdkn1b alleles accelerates spontaneous neoplastic transformation and incidence of tumors of various histological origins. Cell proliferation, but not cell survival, is increased in Pten+/−/Cdkn1b−/− mice. Moreover, Pten+/−/Cdkn1b−/− mice develop prostate carcinoma at complete penetrance within three months from birth. These cancers recapitulate the natural history and pathological features of human prostate cancer. Our findings reveal the crucial relevance of the combined tumor-suppressive activity of Pten and p27Kip1 through the control of cell-cycle progression.

The ETS protein MEF plays a critical role in perforin gene expression and the development of natural killer and NK-T cells

We utilized gene targeting by homologous recombination to define the role that MEF, a transcriptional activating member of the ETS family of transcription factors, plays in lymphopoiesis. MEF-/- mice have a profound reduction in the number of NK-T and NK cells. Purified MEF-/- NK cells cannot lyse tumor cell targets and secrete only minimal amounts of IFNgamma. Perforin protein expression is severely impaired in MEF-deficient NK cells, likely accounting for the lack of tumor cell cytotoxicity. Promoter studies and chromatin immunoprecipitation analyses demonstrate that MEF and not ETS-1 directly regulates transcription of the perforin gene in NK cells. Our results uncover a specific role of MEF in the development and function of NK cells and in innate immunity.

Positive feedback regulation between AKT activation and fatty acid synthase expression in ovarian carcinoma cells.

Activation of AKT and overexpression of fatty acid synthase (FAS) are frequently observed in human ovarian cancer. To explore a possible connection between AKT and FAS, immunohistochemical analyses were conducted on an ovarian cancer tissue microarray, which revealed a significant correlation between phosphorylated AKT (phospho-AKT) and expression of FAS. To investigate the relationship between phospho-AKT and FAS in vitro, a variety of experiments employing a specific phosphatidylinositol 3-OH kinase (PI3K) inhibitor (LY294002), inducible PTEN expression in PTEN-null cells, or AKT1 siRNA demonstrated that phosphatidylinositol-3 kinase (PI3K)/AKT signaling modulates FAS expression. In contrast, inhibition of FAS activity by the drug C75 resulted in downregulation of phospho-AKT and increased cell death. To explore the functional relationship between phospho-AKT and FAS, we used SKOV3, C200, and OVCAR10 ovarian carcinoma cells, which have constitutively active AKT, and OVCAR5 cells, which have very low basal phospho-AKT levels. Treatment with LY294002 abolished AKT activity and potentiated apoptosis induced by FAS inhibitors cerulenin or C75 only in cells with constitutively active AKT, suggesting that constitutive activation of AKT protects against FAS inhibitor-induced cell death. Furthermore, inhibition of FAS activity by cerulenin or C75 resulted in downregulation of phospho-AKT, which preceded the induction of apoptosis. To investigate the relationship between phospho-AKT and FAS in vivo, severe combined immunodeficient mice injected intraperitoneally with SKOV3 cells were treated with C75. Growth of SKOV3 xenografts was markedly inhibited by C75. Analysis of the levels of phospho-AKT and FAS in C75-treated tumors revealed concordant downregulation of phospho-AKT and FAS. Collectively, our findings are consistent with a working model in which AKT activation regulates FAS expression, at least in part, whereas FAS activity modulates AKT activation.

PTEN in Neural Precursor Cells: Regulation of Migration, Apoptosis, and Proliferation

PTEN is a lipid phosphatase, and PTEN mutations are associated with gliomas, macrocephaly, and mental deficiencies. We have used PTEN +/- mice to assess PTENs role in subventricular zone (SVZ) precursor cells. For cultured SVZ neurosphere cells, haploinsufficiency for PTEN increases phosphorylation of Akt and forkhead transcription factor and slightly enhances proliferation. Based on a filter penetration assay, PTEN +/- cells are substantially more migratory and invasive than +/+ cells. The +/- cells also are more resistant to H(2)O(2)-induced apoptosis. Analysis of PTEN +/- and +/+ mice by BrdU labeling reveals no difference in the rate of cell proliferation in the SVZ. Exit of BrdU-labeled cells from the SVZ and radial migration to the outer layers of the olfactory bulb are more rapid for +/- cells. These observations indicate that PTEN regulates SVZ precursor cell function and is particularly important for migration and apoptosis in response to oxidative stress.

Class reunion: PTEN joins the nuclear crew

Several recent reports have brought conclusive evidence that the tumor suppressor PTEN, once considered a strictly cytoplasmic protein, shuttles to the nuclear compartment, where it joins a variety of components of the same pathway it regulates in the cytoplasm, among which PI3K, PDK1 and AKT. In this review, we focus on the growing supporting evidence for an important physiological role of this nuclear pathway and on the role that alteration of this novel regulatory circuit may play during cell transformation.

Pten Loss in the Mouse Thyroid Causes Goiter and Follicular Adenomas: Insights into Thyroid Function and Cowden Disease Pathogenesis

Inactivation and silencing of the tumor suppressor PTEN are found in many different epithelial tumors, including thyroid neoplasia. Cowden Disease patients, who harbor germ-line PTEN mutations, often display thyroid abnormalities, including multinodular goiter and follicular adenomas, and are at increased risk of thyroid cancer. To gain insights into the role PTEN plays in thyroid function and disease, we have generated a mouse strain, in which Cre-mediated recombination is used to specifically delete Pten in the thyrocytes. We found that Pten mutant mice develop diffuse goiter characterized by extremely enlarged follicles, in the presence of normal thyroid-stimulating hormone and T4 hormone levels. Loss of Pten resulted in a significant increase in the thyrocyte proliferative index, which was more prominent in the female mice, and in increased cell density in the female thyroid glands. Surprisingly, goitrogen treatment did not cause a substantial increase of the mutant thyroid size and increased only to some extent the proliferation index of the female thyrocytes, suggesting that a relevant part of the thyroid-stimulating hormone-induced proliferation signals are funneled through the phosphatidylinositol-3-kinase (PI3K)/Akt cascade. Although complete loss of Pten was not sufficient to cause invasive tumors, over two thirds of the mutant females developed follicular adenomas by 10 months of age, showing that loss of Pten renders the thyroid highly susceptible to neoplastic transformation through mechanisms that include increased thyrocyte proliferation. Our findings show that constitutive activation of the PI3K/Akt cascade is sufficient to stimulate continuous autonomous growth and provide novel clues to the pathogenesis of Cowden Disease and sporadic nontoxic goiter.