[Show abstract][Hide abstract] ABSTRACT: Although considerable evidence suggests that in utero arsenic exposure affects children’s health, these data are mainly from areas of the world where groundwater arsenic levels far exceed the World Health Organization limit of 10 μg/L. We, and others, have found that more common levels of in utero arsenic exposure may also impact children’s health. However, the underlying molecular mechanisms are poorly understood. To address this issue, we analyzed the expression of key developmental genes in fetal placenta in a birth cohort of women using unregulated water supplies in a US region with elevated groundwater arsenic. We identified several genes whose expression associated with maternal arsenic exposure in a fetal sex-specific manner. In particular, expression of the HEDGEHOG pathway component, GLI3, in female placentae was both negatively associated with arsenic exposure and positively associated with infant birth weight. This suggests that modulation of GLI3 in the fetal placenta, and perhaps in other fetal tissues, contributes to arsenic’s detrimental effects on fetal growth. We showed previously that arsenic-exposed NIH3T3 cells have reduced GLI3 repressor protein. Together, these studies identify GLI3 as a key signaling node that is affected by arsenic, mediating a subset of its effects on developmental signaling and fetal health.
[Show abstract][Hide abstract] ABSTRACT: Arsenic has wide-ranging effects on human health and there is evidence that it alters the immune response by influencing CD4 +/CD8 + T cell ratios, IL-2 cytokine levels, and the expression of immune-response genes. We investigated the impact of in utero environmental arsenic exposure on immune development and function in newborns participating in a pregnancy cohort in New Hampshire, U.S., where arsenic levels have exceeded the current EPA maximum contaminant level of 10 μg/L. Our results showed that maternal urinary arsenic concentrations were inversely related to absolute total CD45RA + CD4 + cord blood CD69 + T cell counts (N = 116, p = 0.04) and positively associated with CD45RA + CD69- CD294 + cell counts (p = 0.01). In placental samples (N = 70), higher in utero urinary arsenic concentrations were positively associated with expression of IL1β (p = 0.03). These data provide evidence that relatively low-level arsenic exposure in utero may alter the fetal immune system and lead to immune dysregulation.
[Show abstract][Hide abstract] ABSTRACT: Esophageal adenocarcinoma (EAC) ranks sixth in cancer mortality in the world and its incidence has risen dramatically in the western population over the last decades. Data presented herein strongly suggest that Notch signaling is critical for EAC and underlies resistance to chemotherapy. We present evidence that Notch signaling drives a cancer stem cell phenotype by regulating genes that establish stemness. Using patient derived xenograft models we demonstrate that inhibition of Notch by gamma-secretase inhibitors (GSI) is efficacious in downsizing tumor growth. Moreover, we demonstrate that Notch activity in a patient's EUS-derived biopsy might predict outcome to chemotherapy. Therefore, this study provides a proof of concept that inhibition of Notch activity will have efficacy in treating EAC, offering a rationale to lay the foundation for a clinical trial to evaluate the efficacy of GSI in EAC treatment.
Cancer Research 08/2014; 74(21). DOI:10.1158/0008-5472.CAN-14-2051 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mutations in the WNT-pathway regulator ADENOMATOUS POLYPOSIS COLI (APC) promote aberrant activation of the WNT pathway that is responsible for APC-associated diseases such as Familial Adenomatous Polyposis (FAP) and 85% of spontaneous colorectal cancers (CRC). FAP is characterized by multiple intestinal adenomas, which inexorably result in CRC. Surprisingly, given their common occurrence, there are few effective chemotherapeutic drugs for FAP. Here we show that the FDA-approved, anti-helminthic drug Pyrvinium attenuates the growth of WNT-dependent CRC cells and does so via activation of CK1α. Furthermore, we show that Pyrvinium can function as an in vivo inhibitor of WNT-signaling and polyposis in a mouse model of FAP: APCmin mice. Oral administration of Pyrvinium, a CK1α agonist, attenuated the levels of WNT-driven biomarkers and inhibited adenoma formation in APCmin mice. Considering its well-documented safe use for treating enterobiasis in humans, our findings suggest that Pyrvinium could be repurposed for the clinical treatment of APC-associated polyposes.
PLoS ONE 07/2014; 9(7):e101969. DOI:10.1371/journal.pone.0101969 · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Hedgehog (HH) signaling pathway represents an important class of emerging developmental signaling pathways that play critical roles in the genesis of a large number of human cancers. The pharmaceutical industry is currently focused on developing small molecules targeting Smoothened (Smo), a key signaling effector of the HH pathway that regulates the levels and activity of the Gli family of transcription factors. Although one of these compounds vismodegib is now FDA-approved for advanced basal cell carcinoma patients, acquired mutations in Smo can result in rapid relapse. Furthermore, many cancers also exhibit a Smo-independent activation of Gli proteins, an observation that may underlie the limited efficacy of Smo inhibitors in clinical trials against other types of cancer. Thus, there remains a critical need for HH inhibitors with different mechanisms of action, in particularly those that act downstream of Smo. Recently, we identified the FDA-approved anti-pinworm compound pyrvinium as a novel, potent (IC50 ~ 10nM) Casein Kinase-1α (CK1α) agonist. We show here that pyrvinium is a potent inhibitor of HH signaling, which acts by reducing the stability of the Gli family of transcription factors. Consistent with CK1α agonists acting on these most distal components of the HH signaling pathway, pyrvinium is able to inhibit the activity of a clinically relevant, vismodegib resistant Smo mutant, as well as the Gli activity resulting from loss of the negative regulator Suppressor of fused. We go on to demonstrate the utility of this small-molecule in vivo, against the HH dependent cancer medulloblastoma, attenuating its growth and reducing the expression of HH biomarkers.
Cancer Research 07/2014; 74(17). DOI:10.1158/0008-5472.CAN-14-0317 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dysregulation of the hedgehog signaling pathway has been linked to the development and progression of a variety of different human tumors including cancers of the skin, brain, colon, prostate, blood, and pancreas. We assessed the clinicopathological factors that are potentially related to expression of Gli1, the transcription factor that is thought to be the most reliable marker of hedgehog pathway activation in bladder cancer.
[Show abstract][Hide abstract] ABSTRACT: Introduction
Dysregulation of the hedgehog signaling pathway has been linked to the development and progression of a variety of different human tumors including cancers of the skin, brain, colon, prostate, blood, and pancreas. We assessed the clinicopathological factors that are potentially related to expression of Gli1, the transcription factor that is thought to be the most reliable marker of hedgehog pathway activation in bladder cancer.
Bladder cancer cases were identified from the New Hampshire State Cancer Registry as histologically confirmed primary bladder cancer diagnosed between January 1, 2002, and July 31, 2004. Immunohistochemical analysis was performed on a tissue microarray to detect Gli1and p53 expression in these bladder tumors. We computed odds ratios (ORs) and their 95% CIs for Gli1 positivity for pathological category using T category (from TNM), invasiveness, and grade with both the World Health Organization 1973 and World Health Organization International Society of Urological Pathology criteria. We calculated hazard ratios and their 95% CI for Gli1 positivity and recurrence for both Ta-category and invasive bladder tumors (T1+).
A total of 194 men and 67 women, whose tumors were assessable for Gli1 staining, were included in the study. No appreciable differences in Gli1 staining were noted by sex, age, smoking status, or high-risk occupation. Ta-category tumors were more likely to stain for Gli1 as compared with T1-category tumors (adjusted OR = 0.38, CI: 0.17–0.87). Similarly, low-grade (grades 1–2) tumors were more likely to stain for Gli1 as compared with high-grade tumors (grade 3) (adjusted OR = 0.44, CI: 0.21–0.93). In a Cox proportional hazards regression analysis, non–muscle-invasive bladder tumors expressing Gli1 were less likely to recur (adjusted hazard ratio = 0.48; CI: 0.28–0.82; P<0.05) than those in which Gli1 was absent.
Our findings indicate that Gli1 expression may be a marker of low-stage, low-grade bladder tumors and an indicator of a reduced risk of recurrence in this group.
The Journal of Urology 01/2014; DOI:10.1016/j.juro.2012.02.984 · 3.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidemiologic studies and animal models suggest that in utero arsenic exposure affects fetal health, with a negative association between maternal arsenic ingestion and infant birth weight often observed. However, the molecular mechanisms for this association remain elusive. In the present study, we aimed to increase our understanding of the impact of low-dose arsenic exposure on fetal health by identifying possible arsenic-associated fetal tissue biomarkers in a cohort of pregnant women exposed to arsenic at low levels.
Arsenic concentrations were determined from the urine samples of a cohort of 133 pregnant women from New Hampshire. Placental tissue samples collected from enrollees were homogenized and profiled for gene expression across a panel of candidate genes, including known arsenic regulated targets and genes involved in arsenic transport, metabolism, or disease susceptibility. Multivariable adjusted linear regression models were used to examine the relationship of candidate gene expression with arsenic exposure or with birth weight of the baby.
Placental expression of the arsenic transporter AQP9 was positively associated with maternal urinary arsenic levels during pregnancy (coefficient estimate: 0.25; 95% confidence interval: 0.05 -- 0.45). Placental expression of AQP9 related to expression of the phospholipase ENPP2 which was positively associated with infant birth weight (coefficient estimate: 0.28; 95% CI: 0.09 -- 0.47). A structural equation model indicated that these genes may mediate arsenic's effect on infant birth weight (coefficient estimate: -0.009; 95% confidence interval: -0.032 -- -0.001; 10,000 replications for bootstrapping).
We identified the expression of AQP9 as a potential fetal biomarker for arsenic exposure. Further, we identified a positive association between the placental expression of phospholipase ENPP2 and infant birth weight. These findings suggest a path by which arsenic may affect birth outcomes.
Environmental Health 07/2013; 12(1):58. DOI:10.1186/1476-069X-12-58 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The G0/G1 switch gene 2 (G0S2) is rapidly induced by all-trans-retinoic acid (RA)-treatment of acute promyelocytic leukemia (APL) and other cells. G0S2 regulates lipolysis via inhibition of adipose triglyceride lipase (ATGL). This study found that retinoic acid receptor (RAR), but not retinoid X receptor (RXR) agonists induced G0S2 expression in APL cells. Novel G0S2 functions were uncovered that included repression of exogenous gene expression and transcriptional activity. Transient G0S2 transfection repressed the activities of multiple reporter constructs (including the retinoid-regulated species RARβ, UBE1L and G0S2); this occurred in diverse cell contexts. This inhibition was antagonized by siRNA-mediated G0S2 knockdown. To determine the inhibitory effects were not due to transient G0S2 expression, G0S2 was stably overexpressed in cells without appreciable basal G0S2 expression. As expected, this repressed transcriptional activities. Intriguingly, transfection of G0S2 did not affect endogenous RARβ, UBE1L or G0S2 expression. Hence, only exogenously expressed genes were affected by G0S2. The domain responsible for this repression was localized to the G0S2 hydrophobic domain (HD). This was the same region responsible for the ability of G0S2 to inhibit ATGL activity. Whether an interaction with ATGL accounted for this new G0S2 activity was studied. Mimicking the inhibition of ATGL by oleic acid treatment that increased lipid droplet size or ATGL siRNA knockdown did not recapitulate G0S2 repressive effects. Engineered gain of ATGL expression did not rescue G0S2 transcriptional repression either. Thus, transcriptional repression by G0S2 did not depend on the ability of G0S2 to inhibit ATGL. Subcellular localization studies revealed that endogenous and exogenously-expressed G0S2 proteins were localized to the cytoplasm, particularly in the perinuclear region. Expression of a mutant G0S2 species that lacked the HD domain altered cytosolic G0S2 localization. This linked G0S2 subcellular localization to G0S2 transcriptional repression. The potential mechanisms responsible for this G0S2 repression are examined.
International Journal of Oncology 03/2013; 42(5). DOI:10.3892/ijo.2013.1876 · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hedgehog (Hh) proteins regulate the development of a wide range of metazoan embryonic and adult structures, and disruption of Hh signaling pathways results in various human diseases. Here, we provide a comprehensive review of the signaling pathways regulated by Hh, consolidating data from a diverse array of organisms in a variety of scientific disciplines. Similar to the elucidation of many other signaling pathways, our knowledge of Hh signaling developed in a sequential manner centered on its earliest discoveries. Thus, our knowledge of Hh signaling has for the most part focused on elucidating the mechanism by which Hh regulates the Gli family of transcription factors, the so-called "canonical" Hh signaling pathway. However, in the past few years, numerous studies have shown that Hh proteins can also signal through Gli-independent mechanisms collectively referred to as "noncanonical" signaling pathways. Noncanonical Hh signaling is itself subdivided into two distinct signaling modules: (i) those not requiring Smoothened (Smo) and (ii) those downstream of Smo that do not require Gli transcription factors. Thus, Hh signaling is now proposed to occur through a variety of distinct context-dependent signaling modules that have the ability to crosstalk with one another to form an interacting, dynamic Hh signaling network.
[Show abstract][Hide abstract] ABSTRACT: The role of Hedgehog (HH) signaling in bladder cancer remains controversial. The gene encoding the HH receptor and negative regulator PATCHED1 (PTCH1) resides on a region of chromosome 9q, one copy of which is frequently lost in bladder cancer. Inconsistent with PTCH1 functioning as a classic tumor suppressor gene, loss-of-function mutations in the remaining copy of PTCH1 are not commonly found. Here, we provide direct evidence for a critical role of HH signaling in bladder carcinogenesis. We show that transformed human urothelial cells and many urothelial carcinoma cell lines exhibit constitutive HH signaling, which is required for their growth and tumorigenic properties. Surprisingly, rather than originating from loss of PTCH1, the constitutive HH activity observed in urothelial carcinoma cell lines was HH ligand dependent. Consistent with this finding, increased levels of HH and the HH target gene product GLI1 were found in resected human primary bladder tumors. Furthermore, on the basis of the difference in intrinsic HH dependence of urothelial carcinoma cell lines, a gene expression signature was identified that correlated with bladder cancer progression. Our findings therefore indicate that therapeutic targeting of the HH signaling pathway may be beneficial in the clinical management of bladder cancer.
Cancer Research 07/2012; 72(17):4449-58. DOI:10.1158/0008-5472.CAN-11-4123 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Considerable interest has been generated from the results of recent clinical trials using smoothened (SMO) antagonists to inhibit the growth of hedgehog (HH) signaling-dependent tumors. This interest is tempered by the discovery of SMO mutations mediating resistance, underscoring the rationale for developing therapeutic strategies that interrupt HH signaling at levels distinct from those inhibiting SMO function. Here, we demonstrate that HH-dependent non-small cell lung carcinoma (NSCLC) growth is sensitive to blockade of the HH pathway upstream of SMO, at the level of HH ligand processing. Individually, the use of different lentivirally delivered shRNA constructs targeting two functionally distinct HH-processing proteins, skinny hedgehog (SKN) or dispatched-1 (DISP-1), in NSCLC cell lines produced similar decreases in cell proliferation and increased cell death. Further, providing either an exogenous source of processed HH or a SMO agonist reverses these effects. The attenuation of HH processing, by knocking down either of these gene products, also abrogated tumor growth in mouse xenografts. Finally, we extended these findings to primary clinical specimens, showing that SKN is frequently overexpressed in NSCLC and that higher DISP-1 expression is associated with an unfavorable clinical outcome. Our results show a critical role for HH processing in HH-dependent tumors, identifies two potential druggable targets in the HH pathway, and suggest that similar therapeutic strategies could be explored to treat patients harboring HH ligand-dependent cancers.Oncogene advance online publication, 25 June 2012; doi:10.1038/onc.2012.243.
[Show abstract][Hide abstract] ABSTRACT: A number of Smoothened (SMO) pathway antagonists are currently undergoing clinical trials as anticancer agents. These drugs are proposed to attenuate tumor growth solely through inhibition of Hedgehog (HH), which is produced in tumor cells but acts on tumor stromal cells. The pivotal argument underlying this model is that the growth-inhibitory properties of SMO antagonists on HH-producing cancer cells are due to their off-target effects. Here, we show that the tumorigenic properties of such lung cancer cells depend on their intrinsic level of HH activity. Notably, reducing HH signaling in these tumor cells decreases HH target gene expression. Taken together, these results question the dogma that autocrine HH signaling plays no role in HH-dependent cancers, and does so without using SMO antagonists.
Cancer Research 05/2011; 71(13):4454-63. DOI:10.1158/0008-5472.CAN-10-2313 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Exposure to the environmental toxicant arsenic, through both contaminated water and food, contributes to significant health problems worldwide. In particular, arsenic exposure is thought to function as a carcinogen for lung, skin, and bladder cancer via mechanisms that remain largely unknown. More recently, the Hedgehog signaling pathway has also been implicated in the progression and maintenance of these same cancers. Based on these similarities, we tested the hypothesis that arsenic may act in part through activating Hedgehog signaling. Here, we show that arsenic is able to activate Hedgehog signaling in several primary and established tissue culture cells as well as in vivo. Arsenic activates Hedgehog signaling by decreasing the stability of the repressor form of GLI3, one of the transcription factors that ultimately regulate Hedgehog activity. We also show, using tumor samples from a cohort of bladder cancer patients, that high levels of arsenic exposure are associated with high levels of Hedgehog activity. Given the important role Hedgehog signaling plays in the maintenance and progression of a variety of tumors, including bladder cancer, these results suggest that arsenic exposure may in part promote cancer through the activation of Hedgehog signaling. Thus, we provide an important insight into the etiology of arsenic-induced human carcinogenesis, which may be relevant to millions of people exposed to high levels of arsenic worldwide.
Cancer Research 02/2010; 70(5):1981-8. DOI:10.1158/0008-5472.CAN-09-2898 · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The hedgehog (Hh) signalling pathway has an evolutionarily conserved role in patterning fields of cells during metazoan development, and is inappropriately activated in cancer. Hh pathway activity is absolutely dependent on signalling by the seven-transmembrane protein smoothened (Smo), which is regulated by the Hh receptor patched (Ptc). Smo signals to an intracellular multi-protein complex containing the Kinesin related protein Costal2 (Cos2), the protein kinase Fused (Fu) and the transcription factor Cubitus interruptus (Ci). In the absence of Hh, this complex regulates the cleavage of full-length Ci to a truncated repressor protein, Ci75, in a process that is dependent on the proteasome and priming phosphorylations by Protein kinase A (PKA). Binding of Hh to Ptc blocks Ptc-mediated Smo inhibition, allowing Smo to signal to the intracellular components to attenuate Ci cleavage. Because of its homology with the Frizzled family of G-protein-coupled receptors (GPCR), a likely candidate for an immediate Smo effector would be a heterotrimeric G protein. However, the role that G proteins may have in Hh signal transduction is unclear and quite controversial, which has led to widespread speculation that Smo signals through a variety of novel G-protein-independent mechanisms. Here we present in vitro and in vivo evidence in Drosophila that Smo activates a G protein to modulate intracellular cyclic AMP levels in response to Hh. Our results demonstrate that Smo functions as a canonical GPCR, which signals through Galphai to regulate Hh pathway activation.