M. Christine Hollander

PTEN loss in the continuum of common cancers, rare syndromes and mouse models

PTEN is among the most frequently inactivated tumour suppressor genes in sporadic cancer. PTEN has dual protein and lipid phosphatase activity, and its tumour suppressor activity is dependent on its lipid phosphatase activity, which negatively regulates the PI3K–AKT–mTOR pathway. Germline mutations in PTEN have been described in a variety of rare syndromes that are collectively known as the PTEN hamartoma tumour syndromes (PHTS). Cowden syndrome is the best-described syndrome within PHTS, with approximately 80% of patients having germline PTEN mutations. Patients with Cowden syndrome have an increased incidence of cancers of the breast, thyroid and endometrium, which correspond to sporadic tumour types that commonly exhibit somatic PTEN inactivation. Pten deletion in mice leads to Cowden syndrome-like phenotypes, and tissue-specific Pten deletion has provided clues to the role of PTEN mutation and loss in specific tumour types. Studying PTEN in the continuum of rare syndromes, common cancers and mouse models provides insight into the role of PTEN in tumorigenesis and will inform targeted drug development.

Nelfinavir, A lead HIV protease inhibitor, is a broad-spectrum, anticancer agent that induces endoplasmic reticulum stress, autophagy, and apoptosis in vitro and in vivo

Purpose: The development of new cancer drugs is slow and costly. HIV protease inhibitors are Food and Drug Administration approved for HIV patients. Because these drugs cause toxicities that can be associated with inhibition of Akt, an emerging target in cancer, we assessed the potential of HIV protease inhibitors as anticancer agents. Experimental Design: HIV protease inhibitors were screened in vitro using assays that measure cellular proliferation, apoptotic and nonapoptotic cell death, endoplasmic reticulum (ER) stress, autophagy, and activation of Akt. Nelfinavir was tested in non–small cell lung carcinoma (NSCLC) xenografts with biomarker assessment. Results: Three of six HIV protease inhibitors, nelfinavir, ritonavir, and saquinavir, inhibited proliferation of NSCLC cells, as well as every cell line in the NCI60 cell line panel. Nelfinavir was most potent with a mean 50% growth inhibition of 5.2 μmol/L, a concentration achievable in HIV patients. Nelfinavir caused two types of cell death, caspase-dependent apoptosis and caspase-independent death that was characterized by induction of ER stress and autophagy. Autophagy was protective because an inhibitor of autophagy increased nelfinavir-induced death. Akt was variably inhibited by HIV protease inhibitors, but nelfinavir caused the greatest inhibition of endogenous and growth factor–induced Akt activation. Nelfinavir decreased the viability of a panel of drug-resistant breast cancer cell lines and inhibited the growth of NSCLC xenografts that was associated with induction of ER stress, autophagy, and apoptosis. Conclusions: Nelfinavir is a lead HIV protease inhibitor with pleiotropic effects in cancer cells. Given its wide spectrum of activity, oral availability, and familiarity of administration, nelfinavir is a Food and Drug Administration–approved drug that could be repositioned as a cancer therapeutic.

Role of Gadd45 in apoptosis.

gadd45 is a p53-regulated growth arrest and DNA-damage-inducible gene that is also regulated in a p53-independent manner. Whether Gadd45 plays a direct role in apoptosis remains unclear. Microinjection of the exogenous gadd45 expression vector into human fibroblasts has been shown to cause G2 arrest but not apoptosis. Recent studies suggest that Gadd45 may mediate genotoxic stress or Brca1-induced apoptosis via activation of c-Jun N-terminal kinase (JNK) and/or p38 mitogen-activated protein kinase (MAPK). Analyses of gadd45-deficient mice and cells have revealed that Gadd45 appears to exhibit pleiotropic effects, including cell cycle arrest at G2/M, DNA damage repair, and control of genomic stability, but is not required for radiation-induced apoptosis. Furthermore, stress-induced activation of JNK and p38 MAPK is not altered in gadd45-deficient embryonic fibroblasts, suggesting that the lack of Gadd45 may not affect the JNK and p38 MAPK activity. Thus, although the evidence from gadd45-null cells suggests that Gadd45 probably does not play a direct role in genotoxic stress-induced apoptosis, more in-depth studies are needed to firmly establish this contention.

Genotoxic-stress-response genes and growth-arrest genes. gadd, MyD, and other genes induced by treatments eliciting growth arrest.

As discussed throughout this paper, many mammalian DDI genes are associated with growth responses, including both positive responses to growth stimulation and negative responses involving transient growth arrest and terminal differentiation. It is interesting that several immediate-early genes encoding transcription factors, the jun genes, are DDI, are induced by terminal differentiation, and also are associated with positive growth responses. In negative growth-response genes, their control is complex and almost certainly involves multiple regulatory mechanisms. The role of growth-arrest genes after exposure to DNA-damaging agents is currently not known, but as growth arrest can have a protective effect on cells exposed to DNA-damaging agents in both bacteria and eukaryotes, some protective role(s) for the gadd genes may exist. Whatever the roles are for the individual gadd genes, the response of the gadd genes to DNA-damaging agents and other growth-arrest signals has been highly conserved during mammalian evolution, and it is likely that this stress response, as reflected by induction of one or more gadd genes, is present in most or perhaps all mammalian cells. Our findings that the gadd group overlaps with another group of growth-arrest genes, the MyD, indicate that these two groups combined define a new class of genes whose protein products are likely to play a role in cell growth cessation.

Mice Lacking the p53-Effector Gene Gadd45a Develop a Lupus-Like Syndrome

This study addresses the biological function of the p53-effector genes Gadd45a and p21 in the immune system. We find that Gadd45a is a negative regulator of T cell proliferation because, compared to wild-type cells, Gadd45a(-/-) T cells have a lower threshold of activation and proliferate to a greater extent following primary T cell receptor stimulation. Gadd45a(-/-) mice develop an autoimmune disease, similar to human systemic lupus erythematosus (SLE), characterized by high titers of anti-dsDNA, anti-ssDNA, and anti-histone autoantibodies, severe hematological disorders, autoimmune glomerulonephritis, and premature death. Here we show that the lack of both Gadd45a and p21 dramatically accelerates the development of autoimmunity observed in each individual single-gene disruption mutant, demonstrating that these genes play nonredundant roles in the immune response.

Loss of oncogenic H-ras-induced cell cycle arrest and p38 mitogen-activated protein kinase activation by disruption of Gadd45a

ABSTRACT The activation of p53 is a guardian mechanism to protect primary cells from malignant transformation; however, the details of the activation of p53 by oncogenic stress are still incomplete. In this report we show that in Gadd45a −/− mouse embryo fibroblasts (MEF), overexpression of H-ras activates extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) but not p38 kinase, and this correlates with the loss of H-ras-induced cell cycle arrest (premature senescence). Inhibition of p38 mitogen-activated protein kinase (MAPK) activation correlated with the deregulation of p53 activation, and both a p38 MAPK chemical inhibitor and the expression of a dominant-negative p38α inhibited p53 activation in the presence of H-ras in wild-type MEF. p38, but not ERK or JNK, was found in a complex with Gadd45 proteins. The region of interaction was mapped to amino acids 71 to 96, and the central portion (amino acids 71 to 124) of Gadd45a was required for p38 MAPK activation in the presence of H-ras. Our results indicate that this Gadd45/p38 pathway plays an important role in preventing oncogene-induced growth at least in part by regulating the p53 tumor suppressor.

Genomic instability, centrosome amplification, cell cycle checkpoints and Gadd45a

Genomic instability has been a recognized feature of many human tumors for decades. Until recently, however, there was little insight into potential mechanisms for this phenomenon. Recent work has shown first, that increased centrosome numbers (also referred to as centrosome amplification) often accompany genomic instability and second, that when centrosome numbers are increased, cells become genetically unstable. Deletion of Gadd45a leads to centrosome amplification and consequent abnormal mitosis and aneuploidy. Gadd45a is known to be involved in a G2 checkpoint and may be involved in the normal progression from G2 to M and its coordination with S phase events. Whether these functions contribute to prevention of centrosome amplification is being investigated. However, potential mechanisms can be proposed based on known protein associations with Gadd45a, as well as proteins that regulate Gadd45a transcription and are also required for efficient coordination of centrosome duplication and DNA synthesis.

Identification of a Highly Effective Rapamycin Schedule that Markedly Reduces the Size, Multiplicity, and Phenotypic Progression of Tobacco Carcinogen–Induced Murine Lung Tumors

Purpose: Human and murine preneoplastic lung lesions induced by tobacco exposure are characterized by increased activation of the Akt/mammalian target of rapamycin (mTOR) pathway, suggesting a role for this pathway in lung cancer development. To test this, we did studies with rapamycin, an inhibitor of mTOR, in A/J mice that had been exposed to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Experimental Design: Tumorigenesis was induced by i.p. injection of NNK, and rapamycin was administered 1 or 26 weeks after NNK administration. Biomarkers associated with mTOR inhibition were assessed in lung and/or surrogate tissues using immunohistochemistry and immunoblotting. Rapamycin levels were measured using mass spectroscopy. Results: Rapamycin was administered on a daily (5 of 7 days) regimen beginning 26 weeks after NNK decreased tumor size, proliferative rate, and mTOR activity. Multiplicity was not affected. Comparing this regimen with an every-other-day (qod) regimen revealed that rapamycin levels were better maintained with qod administration, reaching a nadir of 16.4 ng/mL, a level relevant in humans. When begun 1 week after NNK, this regimen was well tolerated and decreased tumor multiplicity by 90%. Tumors that did develop showed decreased phenotypic progression and a 74% decrease in size that correlated with decreased proliferation and inhibition of mTOR. Conclusions: Tobacco carcinogen–induced lung tumors in A/J mice are dependent upon mTOR activity because rapamycin markedly reduced the development and growth of tumors. Combined with the Food and Drug Administration approval of rapamycin and broad clinical experience, these studies provide a rationale to assess rapamycin in trials with smokers at high risk to develop lung cancer.

Identification of several human homologs of hamster DNA damage-inducible transcripts. Cloning and characterization of a novel UV-inducible cDNA that codes for a putative RNA-binding protein.

Low ratio hybridization subtraction technique was previously used in this laboratory to enrich and isolate a number of low abundance UV-inducible hamster transcripts (Fornace, A. J., Jr., Alamo, I. J., and Hollander, M. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8800–8804) that led to the identification and cloning of five important hamster and humanGADD genes (Fornace, A. J., Jr., Nebert, D. W., Hollander, M. C., Luethy, J. D., Papathanasiou, M., Fargnoli, J., and Holbrook, N. J. (1989) Mol. Cell. Biol.9, 4196–4203). In this study we have characterized the remaining DNA damage-inducible (DDI) transcripts. Of the 24 DDI clones, 3 clones (A13, A20, and A113) representing different regions of the same hamster cDNA exhibited near perfect homology to human p21 WAF1/CIP1 cDNA. The DDI clones A26, A88, and A99 displayed very high sequence homologies with the human proliferating nuclear antigen, rat translation initiation factor-5 (eIF-5), and human thrombomodulin, respectively, whereas clones A29 and A121 matched with express sequence tagged sequences of unknown identity. The DDI clones A18, 106, and A107 were different isolates of the same hamster cDNA (hereafter referred to as A18) and displayed high sequence homology with the members in the heterogeneous ribonucleoprotein (hnRNP) family. Using the hamster A18 partial-length cDNA as a probe, we screened human fibroblast cDNA library and isolated the corresponding full-length human cDNA. The deduced amino acid sequence revealed that the putative protein contains all the canonical features of a novel glycine-rich hnRNP. The A18 mRNA levels were specifically increased in response to DNA damage induced by UV irradiation or UV mimetic agents. Thus the putative A18 hnRNP is the first hnRNP whose mRNA is specifically regulated in response to UV-induced DNA damage; accordingly, it may play some role in repair of UV-type DNA damage.

Evidence of ultraviolet type mutations in xeroderma pigmentosum melanomas

To look for a direct role of ultraviolet radiation (UV) exposure in cutaneous melanoma induction, we studied xeroderma pigmentosum (XP) patients who have defective DNA repair resulting in a 1000-fold increase in melanoma risk. These XP melanomas have the same anatomic distribution as melanomas in the general population. We analyzed laser capture microdissection samples of skin melanomas from XP patients studied at the National Institutes of Health. The tumor suppressor gene PTEN was sequenced and analyzed for UV-induced mutations. Samples from 59 melanomas (47 melanomas in situ and 12 invasive melanomas) from 8 XP patients showed mutations in the PTEN tumor suppressor gene in 56% of the melanomas. Further, 91% of the melanomas with mutations had 1 to 4 UV type base substitution mutations (occurring at adjacent pyrimidines) (P < 0.0001 compared to random mutations). We found a high frequency of amino-acid-altering mutations in the melanomas and demonstrated that these mutations impaired PTEN function; UV damage plays a direct role in induction of mutations and in inactivation of the PTEN gene in XP melanomas including in situ, the earliest stage of melanoma. This gene is known to be a key regulator of carcinogenesis and therefore these data provide solid mechanistic support for UV protection for prevention of melanoma.