Jun Long

The BET Bromodomain Inhibitor I-BET151 Acts Downstream of Smoothened Protein to Abrogate the Growth of Hedgehog Protein-driven Cancers

Background: Epigenetic regulation plays an important role in cancer-associated signaling pathways. Results: The bromodomain protein inhibitor I-BET151 attenuates Hedgehog signaling downstream of Smoothened. Conclusion: I-BET151 mediated inhibition of Hedgehog-Gli activity acts downstream of Smoothened. Significance: Modulation of bromodomain protein activity attenuates the growth of Hedgehog-driven tumors. 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.

Hedgehog-Producing Cancer Cells Respond to and Require Autocrine Hedgehog Activity

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.

Hedgehog Signaling Regulates Bladder Cancer Growth and Tumorigenicity

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.

The Hedgehog processing pathway is required for NSCLC growth and survival

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.

Identification of a family of fatty-acid-speciated sonic hedgehog proteins, whose members display differential biological properties

Hedgehog (HH) proteins are proteolytically processed into a biologically active form that is covalently modified by cholesterol and palmitate. However, most studies of HH biogenesis have characterized protein from cells in which HH is overexpressed. We purified Sonic Hedgehog (SHH) from cells expressing physiologically relevant levels and showed that it was more potent than SHH isolated from overexpressing cells. Furthermore, the SHH in our preparations was modified with a diverse spectrum of fatty acids on its amino termini, and this spectrum of fatty acids varied dramatically depending on the growth conditions of the cells. The fatty acid composition of SHH affected its trafficking to lipid rafts as well as its potency. Our results suggest that HH proteins exist as a family of diverse lipid-speciated proteins that might be altered in different physiological and pathological contexts in order to regulate distinct properties of HH proteins.

Casein Kinase 1δ Is an APC/CCdh1 Substrate that Regulates Cerebellar Granule Cell Neurogenesis

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/C(Cdh1)) ubiquitin ligase, and conditional deletion of the APC/C(Cdh1) activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/C(Cdh1) also downregulates CK1δ during cell-cycle exit. Therefore, we conclude that APC/C(Cdh1) 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.

Differential abundance of CK1a provides selectivity for pharmacological CK1a activators to target WNT-dependent tumors

A small-molecule CK1α activator inhibits the growth of WNT-driven intestinal tumors without toxicity to normal epithelium. Treating WNT-driven tumors The WNT signaling pathway powers the growth of various tumors, particularly colorectal cancer (CRC). However, WNT-targeted inhibitors are very toxic to normal gastrointestinal tissue, precluding their approval for clinical use. Li et al. show that WNT could be targeted by activating a kinase that inhibits the pathway. A small-molecule activator of the kinase CK1α called SSTC3 suppressed WNT activity in CRC cell lines, prevented tumor growth, and increased survival in mouse models of primary and metastatic CRC. Because the effects of SSTC3 were selective to cells with high WNT activity and low CK1α abundance, SSTC3 was minimally toxic to normal gastrointestinal epithelium. Thus, SSTC3 and its future derivatives may be a promising therapeutic for CRC patients. Constitutive WNT activity drives the growth of various human tumors, including nearly all colorectal cancers (CRCs). Despite this prominence in cancer, no WNT inhibitor is currently approved for use in the clinic largely due to the small number of druggable signaling components in the WNT pathway and the substantial toxicity to normal gastrointestinal tissue. We have shown that pyrvinium, which activates casein kinase 1α (CK1α), is a potent inhibitor of WNT signaling. However, its poor bioavailability limited the ability to test this first-in-class WNT inhibitor in vivo. We characterized a novel small-molecule CK1α activator called SSTC3, which has better pharmacokinetic properties than pyrvinium, and found that it inhibited the growth of CRC xenografts in mice. SSTC3 also attenuated the growth of a patient-derived metastatic CRC xenograft, for which few therapies exist. SSTC3 exhibited minimal gastrointestinal toxicity compared to other classes of WNT inhibitors. Consistent with this observation, we showed that the abundance of the SSTC3 target, CK1α, was decreased in WNT-driven tumors relative to normal gastrointestinal tissue, and knocking down CK1α increased cellular sensitivity to SSTC3. Thus, we propose that distinct CK1α abundance provides an enhanced therapeutic index for pharmacological CK1α activators to target WNT-driven tumors.

Arsenic Attenuates GLI Signaling, Increasing or Decreasing its Transcriptional Program in a Context Dependent Manner

The metalloid arsenic is a worldwide environmental toxicant, exposure to which is associated with many adverse outcomes. Arsenic is also an effective therapeutic agent in certain disease settings. Arsenic was recently shown to regulate the activity of the Hedgehog (HH) signal transduction pathway, and this regulation of HH signaling was proposed to be responsible for a subset of arsenic’s biologic effects. Surprisingly, these separate reports proposed contradictory activities for arsenic, as either an agonist or antagonist of HH signaling. Here we provide in vitro and in vivo evidence that arsenic acts as a modulator of the activity of the HH effector protein glioma-associated oncogene family zinc finger (GLI), activating or inhibiting GLI activity in a context-dependent manner. This arsenic-induced modulation of HH signaling is observed in cultured cells, patients with colorectal cancer who have received arsenic-based therapy, and a mouse colorectal cancer xenograft model. Our results show that arsenic activates GLI signaling when the intrinsic GLI activity is low but inhibits signaling in the presence of high-level GLI activity. Furthermore, we show that this modulation occurs downstream of primary cilia, evidenced by experiments in suppressor of fused homolog (SUFU) deficient cells. Combining our findings with previous reports, we present an inclusive model in which arsenic plays dual roles in GLI signaling modulation: when GLIs are primarily in their repressor form, arsenic antagonizes their repression capacity, leading to low-level GLI activation, but when GLIs are primarily in their activator form, arsenic attenuates their activity.

Inhibition of WNT signaling attenuates self-renewal of SHH-subgroup medulloblastoma

The SMOOTHENED inhibitor vismodegib is FDA approved for advanced basal cell carcinoma (BCC), and shows promise in clinical trials for SONIC HEDGEHOG (SHH)-subgroup medulloblastoma (MB) patients. Clinical experience with BCC patients shows that continuous exposure to vismodegib is necessary to prevent tumor recurrence, suggesting the existence of a vismodegib-resistant reservoir of tumor-propagating cells. We isolated such tumor-propagating cells from a mouse model of SHH-subgroup MB and grew them as sphere cultures. These cultures were enriched for the MB progenitor marker SOX2 and formed tumors in vivo. Moreover, while their ability to self-renew was resistant to SHH inhibitors, as has been previously suggested, this self-renewal was instead WNT-dependent. We show here that loss of Trp53 activates canonical WNT signaling in these SOX2-enriched cultures. Importantly, a small molecule WNT inhibitor was able to reduce the propagation and growth of SHH-subgroup MB in vivo, in an on-target manner, leading to increased survival. Our results imply that the tumor-propagating cells driving the growth of bulk SHH-dependent MB are themselves WNT dependent. Further, our data suggest combination therapy with WNT and SHH inhibitors as a therapeutic strategy in patients with SHH-subgroup MB, in order to decrease the tumor recurrence commonly observed in patients treated with vismodegib.

Abstract LB-430: Hedgehog signaling is required for the survival and tumorigenicity of cancer cells.

Background: Using small molecule smoothened (SMO) antagonists, Hedgehog (HH) signaling had been implicated to play an important role in a variety of cancers (e.g. Brain, Lung, Pancreas, digestive tract etc.). Recently, based on studies in a selected set of pancreatic and colorectal cancer cells it has been suggested: a) earlier observation of HH signaling dependence of various cancers by others and us is due to non-specific effect of SMO antagonists; b) HH producing cancer cells do not have a functional HH pathway; c) HH signaling is not required for the growth or survival of cancer cells rather it affects the growth of tumor by activating HH pathway in stroma cells. Given the reported prevalence of HH signaling in cancers and the suggested implication of this latter work on devising a successful cancer therapy, we decided to investigate the role of HH signaling in cancer in a manner independent of SMO antagonist. Lung cancer, being one of the deadliest cancers with the reported higher incidence of HH signaling, was used as a test system to evaluate the basic assumptions of this recently proposed model of HH role in cancer. Objective: Define the role of HH signaling in cancer cells. Methodology: The human non-small cell lung carcinoma (NSCLC) cell lines (HOP62, A549, U1752, H23, H157, H522) were used as a model system to study the relevance of HH signaling in cancer cells. The HH signaling was modulated by shRNA mediated knockdown of various HH signaling components (e.g. SMO, GLI1, SHH). The lentiviruses expressing shRNA were made and used to deliver the shRNA in NSCLC cells. HH responsiveness (quantitative real time RT-PCR and western blotting), cell proliferation (ATP quantitation), survival (Annexin-V labeling), anchorage independent growth (soft agar assay) and tumorigenesis (xenografts in Nu/Nu nude mice) were evaluated following shRNA mediated attenuation of HH signaling. Currently, HH pathway activation in cancer and stroma cells of primary lung cancer samples is being evaluated by immunohistochemistry (IHC) and RNA in situ hybridization (ISH). Preliminary Results and Conclusions: Exogenous expression of SHH in HOP62 and A549 induced the HH target gene GLI1 and PTCH1 expression while shRNA mediated knockdown of HH pathway components (e.g. SMO, SHH) inhibited the HH target gene expression. Further more, knockdown of HH pathway components, attenuated the proliferation, increased the apoptosis while decreased the anchorage independent growth of tested lung cancer cell lines. GLI1 shRNA mediated attenuation of HH signaling in A549 cells, greatly reduced their tumorigenicity in nude mice. We anticipate seeing activated HH signaling in cancer and stroma cells of primary lung tumor samples by IHC and ISH. Together, these results would indicate that cancer cells do elaborate a functional HH signaling pathway and require HH signaling for growth, survival and tumorigenicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-430. doi:10.1158/1538-7445.AM2011-LB-430