Convergent etiology of Eker rat and human uterine leiomyoma

The Eker rat model has allowed researchers the unique opportunity to study the tumorigenesis of spontaneously occurring uterine leiomyoma. Animals in this line harbor a germline mutation in the tuberous sclerosis complex-2 (Tsc-2) tumor suppressor gene and develop uterine leiomyomas at a rate of ∼65%. Primary leiomyomas obtained from humans and Eker rats along with Eker-derived leiomyoma cell lines were used in studies described herein to determine the effect of PPARγ ligand treatment on the proliferation of this cell type and to determine the role of tuberin and p27Kip1 in the etiology of this tumor type. Treatment of leiomyoma cells of human and rat origin with PPARγ-activating compounds resulted in decreased proliferation. Additionally, PPARγ ligands inhibited estrogen-dependent gene transactivation in Eker-derived leiomyoma cells suggesting that nuclear receptor cross-talk may exist between PPAR and the ER and may be responsible for the inhibition of proliferation in this cell type. Loss of tuberin, the product of the TSC-2 gene, is associated with Eker rat leiomyoma development while the role of this tumor suppressor in human leiomyoma development is unknown. Data herein show that tuberin expression is diminished in 25% of human leiomyomas tested. Additionally, we observed diminished p27 Kip1 expression in 80% of human uterine leiomyomas compared to normal myometrium. Interestingly, the loss of tuberin expression in human leiomyoma was associated with cytoplasmic p27Kip1 accumulation in this cell type. Furthermore, tuberin-null Eker rat leiomyomas and derived cell lines had predominantly cytoplasmic p27Kip1 compared to tuberin-expressing normal myometrium. Taken together, our data show that human and Eker rat leiomyoma proliferation is inhibited upon PPARγ treatment and that the etiology of human and Eker rat leiomyoma converge at loss of p27Kip1 function. Furthermore, our data indicate that the loss of p27 Kip1 function is mediated by loss of expression (in 80% of human leiomyoma) or cytoplasmic localization potentially resulting from the loss of tuberin. ^

Molecular mechanisms of NF-kappaB activation in metastatic pancreatic cancer cells

Pancreatic adenocarcinoma is the fourth leading cause of adult cancer death in the United States. At the time of diagnosis, most patients with pancreatic cancer have advanced and metastatic disease, which makes most of the traditional therapeutic strategies are ineffective for pancreatic cancer. A better understanding of the molecular basis of pancreatic cancer will provide the approach to identify the new strategies for early diagnosis and treatment. NF-κB is a family of transcription factor that play important roles in immune response, cell growth, apoptosis, and tumor development. We have shown that NF-κB is constitutively activated in most human pancreatic tumor tissues and cell lines, but not in the normal tissues and HPV E6E7 gene-immortalized human pancreatic ductal epithelial cells (HPDE/E6E7). By infecting the pancreatic cancer cell line Aspc-1 with a replication defective retrovirus expressing phosphorylation-defective IκBα (IκBαM), the constitutive NF-κB activation is blocked. Subsequent injection of this Aspc-1/IκBαM cells into the pancreas of athymic nude mice showed that liver metastasis is suppressed by the blockade of NF-κB activation. Current studies showed that an autocrine mechanism accounts for the constitutive activation of NF-κB in metastatic human pancreatic cancer cell lines, but not in nonmetastatic human pancreatic cancer cell lines. Further investigation showed that interleukin-1α (IL-1α) was the primary cytokine secreted by these cells that activates NF-κB. Inhibition of IL-1α activity suppressed the constitutive activation of NF-κB and the expression of its downstream target gene, uPA, in metastatic pancreatic cancer cell lines. Even though IL-1α is one of the previously identified NF-κB downstream target genes, our results demonstrate that regulation of IL-1α expression is independent of NF-κB and primarily dependent on AP-1 activity, which is in part induced by overexpression of EGF receptors and activation of MAP kinases. In conclusion, our findings suggest a possible mechanism by which NF-κB is constitutively activated in metastatic human pancreatic cancer cells and a possible missing mechanistic links between inflammation and cancer. ^

Identification of putative 1p36 tumor suppressor genes involved in oligodendroglioma development

Oligodendrogliomas are primary neoplasms of the central nervous system (CNS). One of the most common and characteristic chromosomal abnormalities observed in oligodendroglioma is allelic loss of 1p (Reifenberger et al., 1994; Bello et al., 1995). Since 1p loss has been reported for both well-differentiated and anaplastic oligodendroglioma, it is believed to occur early in tumor development (Bello et al., 1995). This allelic loss also has clinical significance, for oligodendroglioma patients with 1p loss generally respond significantly better to combination chemotherapy and have longer average survival than do oligodendroglioma patients without 1p loss (Cairncross et al., 1998). To date, no genes on 1p have been implicated as essential to the development or treatment response of oligodendroglioma. In order to localize and/or identify a gene involved in oligodendroglioma development, I tested 170 oligodendrogliomas for deletions of 1p and tested 26 tumors for differential expression of genes in the region of 1p36. Evidence obtained from these methods implicated two genes, SHREW1 and the gene encoding DNA fragmentation factor beta (DFFB). The function for the SHREW1 locus is currently not well known, but preliminary data suggests that it a novel member of adherens junctions. The DFFB gene is an enhancer for apoptosis. Thus, both SHREW1 and DFFB may be candidates for an oligodendroglioma tumor suppressor. Mutational analysis of both genes did not uncover any mutations. Future studies will evaluate other mechanisms that may be responsible for inactivation of these genes in oligodendrogliomas. ^

Are functional and genetic components of platelets linked to coronary artery disease: A case-control study

Coronary artery disease (CAD) is a multifactorial disease process involving behavioral, inflammatory, clinical, thrombotic, and genetic components. Previous epidemiologic studies focused on identifying behavioral and demographic risk factors of CAD, but none focused on platelets. Current platelet literature lacks the known effects of platelet function and platelet receptor polymorphisms on CAD. This case-control analysis addressed these issues by analyzing data collected for a previous study. Cases were individuals who had undergone CABG and thus had been diagnosed with CAD, while the controls were volunteers presumed to be CAD free. The platelet function variables analyzed included fibrinogen Von Willebrand Factor activity (VWF), shear-induced platelet aggregation (SIPA), sCD40L, and mean platelet volume; and the platelet polymorphisms studied included PIA, α2 807, Ko, Kozak, and VNTR. Univariate analysis found fibrinogen, VWF, SIPA, and PIA to be independent risk factors of CAD. Logistic regression was used to build a predictive model for CAD using the platelet function and platelet polymorphism data adjusted for age, sex, race, and current smoking status. A model containing only platelet polymorphisms and their respective receptor densities, found polymorphisms within GPIbα to be associated with CAD, yielding an 86% (95% C.I. 0.97–3.55) increased risk with the presence of at least 1 polymorphism in Ko, Kozak, or VNTR. Another model included both platelet function and platelet polymorphism data. Fibrinogen, the receptor density of GPIbα, and the polymorphism in GPIa-IIa (α2 807) were all associated with CAD with odds ratios of 1.10, 1.04, and 2.30 for fibrinogen (10mg/dl increase), GPIbα receptors (1 MFI increase), and GPIa-IIa, respectively. In addition, risk estimates and 99% confidence intervals adjusted for race were calculated to determine if the presence of a platelet receptor polymorphism was associated with CAD. The results were as follows: PIA (1.64, 0.74–3.65); α2 807 (1.35, 0.77–2.37); Ko (1.71, 0.70–4.16); Kozak (1.17, 0.54–2.52); and VNTR (1.24, 0.52–2.91). Although not statistically significant, all platelet polymorphisms were associated with an increased risk for CAD. These exploratory findings indicate that platelets do appear to have a role in atherosclerosis and that anti-platelet drugs targeting GPI-IIa and GPIbα may be better treatment candidates for individuals with CAD. ^

The role of Akt activation in mouse skin tumor promotion

The importance of IGF-1/IGF-1R signaling is evident in human cancers including breast, colon, prostate, and lung which have been shown to overexpress IGF-1. Also, serum levels of IGF-1 have been identified as a risk factor for these cancers. IGF-1 has been primarily shown to mediate its mitogenic effects through signaling pathways such as MAPK and PI3K/Akt. In this regard, BK5.IGF-1 transgenic mice were generated and these mice displayed hyperplasia and hyperkeratosis in the epidermis. In addition, these mice were also found to have elevated MAPK, PI3K, and Akt activities. Furthermore, overexpression of IGF-1 in epidermis can act as a tumor promoter. BK5.IGF-1 transgenic mice developed papillomas after initiation with DMBA without further treatment with a tumor promoter such as TPA. Previous data has also shown that inhibition of the PI3K/Akt signaling pathway by the inhibitor LY294002 was able to reduce the number of tumors formed by IGF-1 mediated tumor promotion. The current studies presented demonstrate that Akt may be the critical effector molecule in IGF-1/IGF-1R mediated tumor promotion. We have found that inhibition of PI3K/Akt by LY294002 inhibits cell cycle components, particularly those associated with G1 to S phase transition including Cyclin D1, Cyclin E, E2F1, and E2F4, that are elevated in epidermis of BK5.IGF-1 transgenic mice. We have also demonstrated that Akt activation may be a central theme in early tumor promotion. In this regard, treatment with diverse tumor promoters such as TPA, okadaic acid, chrysarobin, and UVB was shown to activate epidermal Akt and its downstream signaling pathways after a single treatment. Furthermore, overexpression of Akt targeted to the basal cells of the epidermis led to hyperplasia and increased labeling index as determined by BrdU staining. These mice also had constitutively elevated levels of cell cycle components, particularly Cyclin D1, Cyclin E, E2F1, E2F4, and Mdm-2. These mice developed skin tumors following initiation with DMBA and were hypersensitive to the tumor promoting effects of TPA. Collectively, these studies provide evidence that Akt activation plays an important role in the process of mouse skin tumor promotion. ^

A comprehensive assessment of environmental ultraviolet radiation induced DNA damage in marine microorganisms

There is evidence that ultraviolet radiation (UVR) is increasing over certain locations on the Earth's surface. Of primary concern is the annual pattern of ozone depletion over Antarctica and the Southern Ocean. Reduction of ozone concentration selectively limits absorption of solar UV-B (290–320 nm), resulting in higher irradiance at the Earth's surface. The effects of ozone depletion on the human population and natural ecosystems, particularly the marine environment, are a matter of considerable concern. Indeed, marine plankton may serve as sensitive indicators of ozone depletion and UV-B fluctuations. Direct biological effects of UVR result from absorption of UV-B by DNA. Once absorbed, energy is dissipated by a variety of pathways, including covalent chemical reactions leading to the formation of photoproducts. The major types of photoproduct formed are cyclobutyl pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone dimer [(6-4)PD]. Marine plankton repair these photoproducts using light-dependent photoenzymatic repair or nucleotide excision repair. The studies here show that fluctuations in CPD concentrations in the marine environment at Palmer Station, Antarctica correlate well with ozone concentration and UV-B irradiance at the Earth's surface. A comparison of photoproduct levels in marine plankton and DNA dosimeters show that bacterioplankton display higher resistance to solar UVR than phytoplankton in an ozone depleted environment. DNA damage in marine microorganisms was investigated during two separate latitudinal transects which covered a total range of 140°. We observed the same pattern of change in DNA damage levels in dosimeters and marine plankton as measured using two distinct quantitative techniques. Results from the transects show that differences in photosensitivity exist in marine plankton collected under varying UVR environments. Laboratory studies of Antarctic bacterial isolates confirm that marine bacterioplankton possess differences in survival, DNA damage induction, and repair following exposure to UVR. Results from DNA damage measurements during ozone season, along a latitudinal gradient, and in marine bacterial isolates suggest that changes in environmental UVR correlate with changes in UV-B induced DNA damage in marine microorganisms. Differences in the ability to tolerate UVR stress under different environmental conditions may determine the composition of the microbial communities inhabiting those environments. ^

Molecular markers of human papillomavirus (HPV) infection and epidemiologic risk factors for squamous intraepithelial lesions (SIL) of the cervix

Infection with certain types of HPV is a necessary event in the development of cervical carcinoma; however, not all women who become infected will progress. While much is known about the molecular influence of HPV E6 and E7 proteins on the malignant transformation, little is known about the additional factors needed to drive the process. Currently, conventional cervical screening is insufficient at identifying women who are likely to progress from premalignant lesions to carcinoma. Aneuploidy and chromatin texture from image cytometry have been suggested as quantitative measures of nuclear damage in premalignant lesions and cancer, and traditional epidemiologic studies have identified potential factors to aid in the discrimination of those lesions likely to progress. ^ In the current study, real-time PCR was used to quantitate mRNA expression of the E7 gene in women exhibiting normal epithelium, LSIL, and HSIL. Quantitative cytometry was used to gather information about the DNA index and chromatin features of cells from the same women. Logistic regression modeling was used to establish predictor variables for histologic grade based on the traditional epidemiologic risk factors and molecular markers. ^ Prevalence of mRNA transcripts was lower among women with normal histology (27%) than for women with LSIL (40%) and HSIL (37%) with mean levels ranging from 2.0 to 4.2. The transcriptional activity of HPV 18 was higher than that of HPV 16 and increased with increasing level of dysplasia, reinforcing the more aggressive nature of HPV 18. DNA index and mRNA level increased with increasing histological grade. Chromatin score was not correlated with histology but was higher for HPV 18 samples and those with both HPV 18 and HPV 16. However, chromatin score and DNA index were not correlated with mRNA levels. The most predictive variables in the regression modeling were mRNA level, DNA index, parity, and age, and the ROC curves for LSIL and HSIL indicated excellent discrimination. ^ Real-time PCR of viral transcripts could provide a more efficient method to analyze the oncogenic potential within cells from cervical swabs. Epidemiological modeling of malignant progression in the cervix should include molecular markers, as well as the traditional epidemiological risk factors. ^

RNA polymerase II large subunit degradation induced by ecteinascidin 743: Molecular characterization and subsequent rational investigation of antitumor mechanisms in cancers with clinical response

Ecteinascidin 743 (Et-743), which is a novel DNA minor groove alkylator with a unique spectrum of antitumor activity, is currently being evaluated in phase II/III clinical trials. Although the precise molecular mechanisms responsible for the observed antitumor activity are poorly understood, recent data suggests that post-translational modifications of RNA polymerase II Large Subunit (RNAPII LS) may play a central role in the cellular response to this promising anticancer agent. The stalling of an actively transcribing RNAPII LS at Et-743-DNA adducts is the initial cellular signal for transcription-coupled nucleotide excision repair (TC-NER). In this manner, Et-743 poisons TC-NER and produces DNA single strand breaks. Et-743 also inhibits the transcription and RNAPII LS-mediated expression of selected genes. Because the poisoning of TC-NER and transcription inhibition are critical components of the molecular response to Et-743 treatment, we have investigated if changes in RNAPII LS contribute to the disruption of these two cellular pathways. In addition, we have studied changes in RNAPII LS in two tumors for which clinical responses were reported in phase I/II clinical trials: renal cell carcinoma and Ewing's sarcoma. Our results demonstrate that Et-743 induces degradation of the RNAPII LS that is dependent on active transcription, a functional 26S proteasome, and requires functional TC-NER, but not global genome repair. Additionally, we have provided the first experimental data indicating that degradation of RNAPII LS might lead to the inhibition of activated gene transcription. A set of studies performed in isogenic renal carcinoma cells deficient in von Hippel-Lindau protein, which is a ubiquitin-E3-ligase for RNAPII LS, confirmed the central role of RNAPII LS degradation in the sensitivity to Et-743. Finally, we have shown that RNAPII LS is also degraded in Ewing's sarcoma tumors following Et-743 treatment and provide data to suggest that this event plays a role in decreased expression of the Ewing's sarcoma oncoprotein, EWS-Fli1. Altogether, these data implicate degradation of RNAPII LS as a critical event following Et-743 exposure and suggest that the clinical activity observed in renal carcinoma and Ewing's sarcoma may be mediated by disruption of molecular pathways requiring a fully functional RNAPII LS. ^

Tackling ErbB2: ErbB2-targeting peptide therapy and combination therapy with trastuzumab plus PI3K inhibitors

ErbB2 is an excellent target for cancer therapies because its overexpression was found in about 30% of breast cancers and correlated with poor prognosis of the patients. Unfortunately, current therapies for ErbB2-positive breast cancers remain unsatisfying due to side effects and resistance, and new therapies for ErbB2 overexpressing breast cancers are needed. Peptide/protein therapy using cell-penetrating peptides (CPPs) as carriers is promising because the internalization is highly efficient and the cargos can be bioactive. The major obstacle in using CPPs for therapy is their lack of specificity. We sought to develop a peptide carrier specifically introducing therapeutics to ErbB2-overexpressing breast cancer cells. By modifying the TAT-derived CPP, and attaching anti-HER2/neu peptide mimetic (AHNP), we developed the peptide carrier (P3-AHNP) specifically targeted ErbB2-overexpressing breast cancers in vitro and in vivo. A STAT3 SH2 domain-binding peptide conjugated to this peptide carrier (P3-AHNP-STAT3BP) was delivered preferentially into ErbB2-overexpressing breast cancer cells in vitro and in vivo. P3-AHNP-STAT3BP inhibited growth and induced apoptosis in vitro, with ErbB2-overexpressing 435.eB cells being more sensitive than the ErbB2-lowexpressing MDA-MB-435 cells. P3-AHNP-STAT3BP preferentially accumulated and inhibited growth in 435.eB xenografts, comparing with MDA-MB-435 xenografts or normal tissues with low levels of ErbB2. This ErbB2-targeting peptide delivery system provided the basis for future development of novel cancer target-specific treatments with low toxicity to normal cells. ^ Another urgent issue in treating ErbB2-positive breast cancers is trastuzumab resistance. Trastuzumab is the only FDA-approved ErbB2-targeting antibody for treatment of metastatic breast cancers overexpressing ErbB2, and has remarkable therapeutic efficacy in certain patients. The overall trastuzumab response rate, however, is limited, and understanding the mechanisms of trastuzumab resistance is needed to overcome this problem. We report that PTEN activation contributes to trastuzumab's anti-tumor activity. Trastuzumab treatment quickly inactivated Src, which reduced PTEN tyrosine phosphorylation, increased PTEN membrane localization and its phosphatase activity in cancer cells. Reducing PTEN expression in breast cancer cells by antisense oligonucleotides conferred trastuzumab resistance in vitro and in vivo. Importantly, PI3K inhibitors sensitized PTEN-deficient breast cancers to the growth inhibition by trastuzumab in vitro and in vivo, suggesting that combination therapies with PI3K inhibitors plus trastuzumab could overcome trastuzumab resistance. ^

Targeted cancer diagnosis with radiolabeled endostatin

Nuclear imaging is used for non-invasive detection, staging and therapeutic monitoring of tumors through the use of radiolabeled probes. Generally, these probes are used for applications in which they provide passive, non-specific information about the target. Therefore, there is a significant need for actively-targeted radioactive probes to provide functional information about the site of interest. This study examined endostatin, an endogenous inhibitor of tumor angiogenesis, which has affinity for tumor vasculature. The major objective of this study was to develop radiolabeled analogues of endostatin through novel chemical and radiochemical syntheses, and to determine their usefulness for tumor imaging using in vitro and in vivo models of vascular, mammary and prostate tumor cells. I hypothesize that this binding will allow for a non-invasive approach to detection of tumor angiogenesis, and such detection can be used for therapeutic monitoring to determine the efficacy of anti-angiogenic therapy. ^ The data showed that endostatin could be successfully conjugated to the bifunctional chelator ethylenedicysteine (EC), and radiolabeled with technetium-99m and gallium-68, providing a unique opportunity to use a single precursor for both nuclear imaging modalities: 99mTc for single photon emission computed tomography and 68Ga for positron emission tomography, respectively. Both radiolabeled analogues showed increased binding as a function of time in human umbilical vein endothelial cells and mammary and prostate tumor cells. Binding could be blocked in a dose-dependent manner by unlabeled endostatin implying the presence of endostatin receptors on both vascular and tumor cells. Animal biodistribution studies demonstrated that both analogues were stable in vivo, showed typical reticuloendothelial and renal excretion and produced favorable absorbed organ doses for application in humans. The imaging data provide evidence that the compounds quantitate tumor volumes with clinically-useful tumor-to-nontumor ratios, and can be used for treatment follow-up to depict changes occurring at the vascular and cellular levels. ^ Two novel endostatin analogues were developed and demonstrated interaction with vascular and tumor cells. Both can be incorporated into existing nuclear imaging platforms allowing for potential wide-spread clinical benefit as well as serving as a diagnostic tool for elucidation of the mechanism of action of endostatin. ^

Adiposity and the perilipin gene: The role of single locus and multilocus variation, obesity-related quantitative traits, and disease-related phenotypes in the Atherosclerosis Risk in Communities (ARIC) study

Obesity is a complex multifactorial disease and is a public health priority. Perilipin coats the surface of lipid droplets in adipocytes and is believed to stabilize these lipid bodies by protecting triglyceride from early lipolysis. This research project evaluated the association between genetic variation within the human perilipin (PLIN) gene and obesity-related quantitative traits and disease-related phenotypes in Non-Hispanic White (NHW) and African American (AA) participants from the Atherosclerosis Risk in Communities (ARIC) Study. ^ Multivariate linear regression, multivariate logistic regression, and Cox proportional hazards models evaluated the association between single gene variants (rs2304794, rs894160, rs8179071, and rs2304795) and multilocus variation (rs894160 and rs2304795) within the PLIN gene and both obesity-related quantitative traits (body weight, body mass index [BMI], waist girth, waist-to-hip ratio [WHR], estimated percent body fat, and plasma total triglycerides) and disease-related phenotypes (prevalent obesity, metabolic syndrome [MetS], prevalent coronary heart disease [CHD], and incident CHD). Single variant analyses were stratified by race and gender within race while multilocus analyses were stratified by race. ^ Single variant analyses revealed that rs2304794 and rs894160 were significantly related to plasma triglyceride levels in all NHWs and NHW women. Among AA women, variant rs8179071 was associated with triglyceride levels and rs2304794 was associated with risk-raising waist circumference (>0.8 in women). The multilocus effects of variants rs894160 and rs2304795 were significantly associated with body weight, waist girth, WHR, estimated percent body fat, class II obesity (BMI ≥ 35 kg/m2), class III obesity (BMI ≥ 35 kg/m2), and risk-raising WHR (>0.9 in men and >0.8 in women) in AAs. Variant rs2304795 was significantly related to prevalent MetS among AA males and prevalent CHD in NHW women; multilocus effects of the PLIN gene were associated with prevalent CHD among NHWs. Rs2304794 was associated with incident CHD in the absence of the MetS among AAs. These findings support the hypothesis that variation within the PLIN gene influences obesity-related traits and disease-related phenotypes. ^ Understanding these effects of the PLIN genotype on the development of obesity can potentially lead to tailored health promotion interventions that are more effective. ^

p16-induced apoptosis in Ewing's sarcoma

The tumor suppressor p16 is a negative regulator of the cell cycle, and acts by preventing the phosphorylation of RB, which in turn prevents the progression from G1 to S phase of the cell cycle. In addition to its role in the cell cycle, p16 may also be able to induce apoptosis in some tumors. Ewing's sarcoma, a pediatric cancer of the bone and soft tissue, was used to study the ability of p16 to induce apoptosis due to the fact that p16 is often deleted in Ewing's sarcoma tumors and may play a role in the oncogenesis or progression of this disease. The purpose of these studies was to determine whether introduction of p16 into Ewing's sarcoma cells would induce apoptosis. We infected the Ewing's sarcoma cell line TC71, which does not express p16, with adenovirus- p16 (Ad-p16). Ad-p16 infection led to the production of functional p16 as measured by the induction of G1 arrest. Ad-p16 infection induced as much as a 100% increase in G1 arrest compared to untreated cells. As measured by propidium iodide (PI) and Annexin V staining, Ad-p16 was able to induce apoptosis to levels 20–30 fold higher than controls. Furthermore, Ad-p16 infection led to loss of RB protein before apoptosis could be detected. The loss of RB protein was due to post-translational degradation of RB, which was inhibited by the addition of the proteasome inhibitors PS-341 and NPI-0052. Downregulation of RB with si-RNA sensitized cells to Ad-p16-induced apoptosis, indicating that RB protects from apoptosis in this model. This study shows that p16 leads to the degradation of RB by the ubiquitin/proteasome pathway, and that this degradation may be important for the induction of apoptosis. Given that RB may protect from apoptosis in some tumors, apoptosis-inducing therapies may be enhanced in tumors which have lost RB expression, or in which RB is artificially inactivated. ^

Effect of thiazolidinedione, a class of anti-diabetic drugs, on the mouse skin tumorigenesis

Thiazolidinediones (TZDs), a novel class of anti-diabetic drugs, have been known as ligands of peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor that belongs to the nuclear receptor superfamily. These synthetic compounds improve insulin sensitivity in patients with type II diabetes likely through activating PAPRγ. Interestingly, they were also shown to inhibit cell growth and proliferation in a wide variety of tumor cell lines. The aim of this study is to assess the potential use of TZDs in the prevention of carcinogenesis using mouse skin as a model. ^ We found that troglitazone, one of TZD drugs, strongly inhibited cultured mouse skin keratinocyte proliferation as demonstrated by [3H]thymidine incorporation assay. It also induced a cell cycle G1 phase arrest and inhibited expression of cell cycle proteins, including cyclin D1, cdk2 and cdk4. Further experiments showed that PPARγ expression in keratinocytes was surprisingly undetectable in vitro or in vivo. Consistent with this, no endogenous PPARγ function in keratinocytes was found, suggesting that the inhibition of troglitazone on keratinocyte proliferation and cell cycle was PPARγ-independent. We further found that troglitazone inhibited insulin/insulin growth factor I (IGF-1) mitogenic signaling, which may explains, at least partly, its inhibitory effect on keratinocyte proliferation. We showed that troglitazone rapidly inhibited IGF-1 induced phosphorylation of p70S6K by mammalian target of rapamycin (mTOR). However, troglitazone did not directly inhibit mTOR kinase activity as shown by in vitro kinase assay. The inhibition of p70S6K is likely to be the result of strong activation of AMP activated protein kinase (AMPK) by TZDs. Stable expression of a dominant negative AMPK in keratinocytes blocked the inhibitory effect of troglitazone on IGF-1 induced phosphorylation of p70S6K. ^ Finally, we found that dietary TZDs inhibited by up to 73% mouse skin tumor development promoted by elevated IGF-1 signaling in BK5-IGF-1 transgenic mice, while they had no or little effect on skin tumor development promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet (UV). Since IGF-1 signaling is frequently found to be elevated in patients with insulin resistance and in many human tumors, our data suggest that TZDs may provide tumor preventive benefit particularly to these patients. ^

The biologic and signaling effects of Tiam1 overexpression in colorectal carcinoma

Cellular migration is essential to many normal cellular processes. In tumor cells, aberrant activation of the normal pathways regulating migration is one of the critical steps in the development of metastasis. Previously, I demonstrated for the first time that overexpression of Tiam1, a guanine nucleotide exchange factor (GNEF) for small G proteins in the Rho family, could alter migration in colorectal tumor cells. ^ This dissertation focuses on the roles of Tiam1 in promoting cell migration, survival, and metastasis of colorectal carcinoma cells, utilizing the model system I developed. To determine the in vivo phenotype of the migratory cell lines, athymic nude mice were injected with cells into the orthotopic site. Several of the mice injected with cells of increased migratory potential had metastases. Thus, the in vitro selection for increased migration resulted in increased metastatic potential in vivo, and therefore, the Tiam1-overexpressing cells provide a model to examine signal transduction pathways important to this process. ^ To examine effects of Tiam1 signaling on small G proteins critical to cellular functions associated with migration, I examined the activation status of the small G proteins Rac, Rho, and Cdc42. The cells of increased migratory potential have increased GTP-bound Rac and Rho, compared to control SW480 cells. Cells that overexpress Tiam1 are more migratory and are resistant to detachment-induced death, or anoikis. To determine which effects and phenotypes were Tiam1-specific, we utilized siRNA to downregulate Tiam1 expression. These results demonstrate that Tiam1 is sufficient but not required for the migration of colorectal carcinoma cells in our model system, and that the biologically selected cells have additional changes that promote migration besides the increase in Tiam1. I also show that Tiam1 protects colorectal carcinoma cells from detachment-induced death, but is not required for anoikis resistance in the biologically selected migratory cells. ^ In summary, my studies demonstrate a heretofore-unknown regulator of phenotypes critical to the development of colorectal carcinoma metastases, overexpression of Tiam1. Understanding the mechanism by which Tiam1 contributes to cellular migration and metastasis is crucial to developing desperately needed new therapies for colorectal carcinoma. ^

Nonlinear tests for genetic studies of complex disease

Linkage and association studies are major analytical tools to search for susceptibility genes for complex diseases. With the availability of large collection of single nucleotide polymorphisms (SNPs) and the rapid progresses for high throughput genotyping technologies, together with the ambitious goals of the International HapMap Project, genetic markers covering the whole genome will be available for genome-wide linkage and association studies. In order not to inflate the type I error rate in performing genome-wide linkage and association studies, multiple adjustment for the significant level for each independent linkage and/or association test is required, and this has led to the suggestion of genome-wide significant cut-off as low as 5 × 10 −7. Almost no linkage and/or association study can meet such a stringent threshold by the standard statistical methods. Developing new statistics with high power is urgently needed to tackle this problem. This dissertation proposes and explores a class of novel test statistics that can be used in both population-based and family-based genetic data by employing a completely new strategy, which uses nonlinear transformation of the sample means to construct test statistics for linkage and association studies. Extensive simulation studies are used to illustrate the properties of the nonlinear test statistics. Power calculations are performed using both analytical and empirical methods. Finally, real data sets are analyzed with the nonlinear test statistics. Results show that the nonlinear test statistics have correct type I error rates, and most of the studied nonlinear test statistics have higher power than the standard chi-square test. This dissertation introduces a new idea to design novel test statistics with high power and might open new ways to mapping susceptibility genes for complex diseases. ^