[Show abstract][Hide abstract] ABSTRACT: Quantitative analysis and modeling of signaling pathway components can reveal important information about the dynamics of that system, including the relative stoichiometries and affinities between the individual signaling components, as well as rate-limiting steps in the signaling pathway. In this chapter, we present a method that we developed to quantify the steady-state ratio of core Hedgehog (Hh) signaling components in both cultured cells and the Drosophila embryo, a physiologically relevant tissue.
[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: Although casein kinase 1δ (CK1δ) is at the center of multiple signaling pathways, its role in the expansion of CNS progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs) as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/CCdh1) ubiquitin ligase, and conditional deletion of the APC/CCdh1 activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/CCdh1 also downregulates CK1δ during cell-cycle exit. Therefore, we conclude that APC/CCdh1 controls CK1δ levels to balance proliferation and cell-cycle exit in the developing CNS. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a therapeutic target.
[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: Epigenetic enzymes modulate signal transduction pathways in different biological contexts. We reasoned that epigenetic regulators
might modulate the Hedgehog (HH) signaling pathway, a main driver of cell proliferation in various cancers including medulloblastoma.
To test this hypothesis, we performed an unbiased small-molecule screen utilizing an HH-dependent reporter cell line (Light2
cells). We incubated Light2 cells with small molecules targeting different epigenetic modulators and identified four histone
deacetylase inhibitors and a bromodomain and extra terminal domain (BET) protein inhibitor (I-BET151) that attenuate HH activity.
I-BET151 was also able to inhibit the expression of HH target genes in Sufu−/− mouse embryonic fibroblasts, in which constitutive Gli activity is activated in a Smoothened (Smo)-independent fashion, consistent
with it acting downstream of Smo. Knockdown of Brd4 (which encodes one of the BET proteins) phenocopies I-BET151 treatment, suggesting that Brd4 is a regulator of the HH signaling
pathway. Consistent with this suggestion, Brd4 associates with the proximal promoter region of the Gli1 locus, and does so in a manner that can be reversed by I-BET151. Importantly, I-BET151 also suppressed the HH activity-dependent
growth of medulloblastoma cells, in vitro and in vivo. These studies suggest that BET protein modulation may be an attractive therapeutic strategy for attenuating the growth of
HH-dependent cancers, such as medulloblastoma.
Journal of Biological Chemistry 10/2014; DOI:10.1074/jbc.M114.595348 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BRCA1 mutation carriers are predisposed to developing basal-like breast cancers with high metastasis and poor prognosis. Yet how BRCA1 suppresses formation of basal-like breast cancers is still obscure. Deletion of p18Ink4c (p18), an inhibitor of CDK4 and CDK6, functionally inactivates the RB pathway, stimulates mammary luminal stem cell proliferation, and leads to spontaneous luminal tumor development. Alternately, germline mutation of Brca1 shifts the fate of luminal cells to cause luminal-to-basal mammary tumor transformation. Here we report that disrupting Brca1 by either germline or epithelium-specific mutation in p18-deficient mice activates epithelial-to-mesenchymal transition (EMT) and induces dedifferentiation of luminal stem cells (LSCs), which associate closely with expansion of basal and cancer stem cells and formation of basal-like tumors. Mechanistically, BRCA1 bound to the TWIST promoter, suppressing its activity and inhibiting EMT in mammary tumor cells. In human luminal cancer cells, BRCA1 silencing was sufficient to activate TWIST and EMT and increase tumor formation. In parallel, TWIST expression and EMT features correlated inversely with BRCA1 expression in human breast cancers. Together, our findings showed that BRCA1 suppressed TWIST and EMT, inhibited LSC dedifferentiation and repressed expansion of basal stem cells and basal-like tumors. Thus, our work offers the first genetic evidence that Brca1 directly suppresses EMT and LSC de-differentiation during breast tumorigenesis.
Cancer Research 09/2014; 74(21). DOI:10.1158/0008-5472.CAN-14-1119 · 9.33 Impact Factor
[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 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Notch signaling pathway governs many distinct cellular processes by regulating transcriptional programs. The transcriptional response initiated by Notch is highly cell context dependent, indicating that multiple factors influence Notch target gene selection and activity. However, the mechanism by which Notch drives target gene transcription is not well understood. Herein, we identify and characterize a novel Notch-interacting protein, NACK, which acts as a Notch transcriptional co-activator. We show that NACK associates with the Notch transcriptional activation complex on DNA, mediates Notch transcriptional activity, and is required for Notch-mediated tumorigenesis. We demonstrate that Notch1 and NACK are co-expressed during mouse development and that homozygous loss of NACK is embryonic lethal. Finally, we show that NACK is also a Notch target gene, establishing a feed forward loop. Thus, our data indicate that NACK is a key component of the Notch transcriptional complex and is an essential regulator of Notch-mediated tumorigenesis and development.
Cancer Research 07/2014; DOI:10.1158/0008-5472.CAN-14-1547 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oncogenic RAS promotes production of reactive oxygen species (ROS), which mediate pro-malignant signaling but can also trigger DNA damage-induced tumor suppression. Thus RAS-driven tumor cells require redox-protective mechanisms to mitigate the damaging aspects of ROS. Here, we show that MutT Homolog 1 (MTH1), the mammalian 8-oxodGTPase that sanitizes oxidative damage in the nucleotide pool, is important for maintaining several KRAS-driven pro-malignant traits in a nonsmall cell lung carcinoma (NSCLC) model. MTH1 suppression in KRAS-mutant NSCLC cells impairs proliferation and xenograft tumor formation. Furthermore, MTH1 levels modulate KRAS-induced transformation of immortalized lung epithelial cells. MTH1 expression is upregulated by oncogenic KRAS and correlates positively with high KRAS levels in NSCLC human tumors. At a molecular level, in p53-competent KRAS-mutant cells, MTH1 loss provokes DNA damage and induction of oncogene-induced senescence. In p53-nonfunctional KRAS-mutant cells, MTH1 suppression does not produce DNA damage but reduces proliferation and leads to an adaptive decrease in KRAS expression levels. Thus, MTH1 not only enables evasion of oxidative DNA damage and its consequences, but can also function as a molecular rheostat for maintaining oncogene expression at optimal levels. Accordingly, our results indicate MTH1 is a novel and critical component of oncogenic KRAS-associated malignancy and its inhibition is likely to yield significant tumor-suppressive outcomes in KRAS-driven tumors.
[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 · 3.23 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 · 9.33 Impact Factor
[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 Gli1 and 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.