PhD Student - Centre for Cancer Research, Hudson Institute of Medical Research, Melbourne, Australia
p53 and Rb, Potential Predictive Biomarkers for the Treatment of Hedgehog-driven Cancers
Cover Image - Neural progenitor cells in an embryonic mouse spinal cord dependent on Hedgehog Signalling for development
The Hedgehog (Hh) signalling pathway plays a critical role in biological processes that occurs in fetal development, such as stem cell maintenance, cell to cell interactions and tissue development. Secreted Hh proteins, termed Hh ligands, activate the pathway in a concentration and time dependent manner, by initiating a series of cellular responses that promotes organ development. The processes of brain, bone, heart, testis and limb development all require Hh pathway activity for successful growth and function. In addition to Hh’s multifaceted roles during normal development, aberrant Hh signalling is responsible for the initiation of progression of various cancers. Overactive Hh signalling has been established in basal cell carcinoma, medulloblastoma, lung, prostate, ovarian and breast cancer, to name a few, making the Hh pathway an area of intense study in both developmental and cancer biology. Because tumour growth and malignancy depend on Hh signalling in various cancers, drugs that block the Hh pathway have been developed to treat Hh cancers. However, recent entry of Hh-inhibitors into clinical trials have revealed mixed patient responses and prompts further exploration of Hh pathway activation, inhibition and resistance to treatment.
Cancer biomarkers, an identifiable or measurable biological molecule that is linked to disease, has made a large impact in cancer research, particularly in cancer diagnosis and how well a patient will respond to a treatment. In Hh-driven cancers, therapeutic translation of Hh-inhibitors has been impeded by lack of predictive genetic biomarkers. Loss or inactivation of the genes p53 and Rb is a common feature in Hh-cancers, where dual loss occurs in over 80% of patients. Although the link between p53 and Rb loss in Hh cancers has been clearly established, a direct association has not been investigated. From inactivating the genes p53 and Rb in various Hh research models of development and cancer, overactivation of the Hh pathway is observed. From pharmacologically blocking the pathway with Hh-inhibitor drugs, upon loss of p53 and Rb, tumours stop growing in lab mice and prolong their survival. Our data implicates p53 and Rb as genetic biomarkers for increased Hh signalling in cancer and potential responsiveness to Hh-inhibitor therapy in patients.
Abstract: Drug resistance poses a great challenge to targeted cancer therapies. In Hedgehog pathway-dependent cancers, the scope of mechanisms enabling resistance to Smo-inhibitors is not known. Here, we performed a transposon mutagenesis screen in medulloblastoma and identified multiple modes of resistance. Surprisingly, mutations in ciliogenesis genes represent a frequent cause of resistance, and patient datasets indicate that cilia loss constitutes a clinically relevant category of resistance. Conventionally, primary cilia are thought to enable oncogenic Hedgehog signaling. Paradoxically, we find that cilia loss protects tumor cells from susceptibility to Smo-inhibitors and maintains a "persister" state that depends on continuous low output of the Hedgehog program. Persister cells can serve as a reservoir for further tumor evolution, as additional alterations synergize with cilia loss to generate aggressive recurrent tumors. Together, our findings reveal novel patterns of resistance and provide mechanistic insights for the role of cilia in tumor evolution and drug resistance.
Pub.: 20 Sep '17, Pinned: 12 Oct '17
Abstract: Astrocytes, the most abundant type of glial cells in the brain, play critical roles in supporting neuronal development and brain function. While astrocytes have been frequently detected in brain tumors, including medulloblastoma (MB), their functions in tumorigenesis are not clear. Here we demonstrate that astrocytes are essential components of the MB tumor microenvironment. Tumor-associated astrocytes (TAA) secreted the ligand sonic hedgehog (Shh), which is required for maintaining MB cell proliferation despite the absence of its primary receptor Patched-1 (Ptch1). Shh drove expression of Nestin in MB cells through a Smoothened-dependent, Gli1-independent mechanism. Ablation of TAA dramatically suppressed Nestin expression and blocked tumor growth. These findings demonstrate an indispensable role for astrocytes in MB tumorigenesis and reveal a novel Ptch1-independent Shh pathway involved in MB progression.
Pub.: 08 Oct '17, Pinned: 12 Oct '17
Abstract: The immunoglobulin superfamily adhesion molecule close homolog of L1 (CHL1) plays important roles during nervous system development. Here, we identified the hedgehog receptor patched-1 (PTCH1) as novel CHL1 binding partner and showed that CHL1 interacts with the first extracellular loop of PTCH1 via its extracellular domain. Co-localization and -immunoprecipitation of CHL1 with PTCH1 suggest an association of CHL1 with this major component of the hedgehog signaling pathway. The trans-interaction of CHL1 with PTCH1 promotes neuronal survival in cultures of dissociated cerebellar granule cells and of organotypic cerebellar slices. An inhibitor of the PTCH1-regulated hedgehog signal transducer smoothened (SMO) and inhibitors of RhoA and Rho-associated kinase (ROCK) prevent CHL1-dependent survival of cultured cerebellar granule cells and survival of cerebellar granule and Purkinje cells in organotypic cultures. In histological sections from 10- and 14-day-old CHL1-deficient mice enhanced apoptosis of granule, but not Purkinje cells was observed. The results of the present study indicate that CHL1 triggers PTCH1-, SMO-, RhoA- and ROCK-dependent signal transduction pathways to promote neuronal survival after cessation of the major morphogenetic events in mouse cerebellar development.
Pub.: 21 Jun '17, Pinned: 12 Oct '17
Abstract: Aberrant Sonic Hedgehog/Gli (Hh/Gli) signaling pathway is a critical regulator of Sonic hedgehog medulloblastoma (SHH-MB). Cancer stem cells (CSCs), thought to be largely responsible for tumor initiation, maintenance, dissemination and relapse, have been identified in SHH-MB. Since we previously demonstrated that Hh/Gli signaling controls CSCs features in SHH-MB and that in these tumors miR-326 is down regulated, here we investigated whether there is a functional link between Hh/Gli signaling and miR-326.We evaluated β-arrestin1 (Arrb1) and its intragenic miR-326 levels in CSCs derived from SHH-MB. Subsequently, we modulated the expression of Arrb1 and miR-326 in CSCs in order to gain insight into their biological role. We also analyzed the mechanism by which Arrb1 and miR-326 control Hh/Gli signaling and self-renewal, using luciferase and protein immunoprecipitation assays.Low levels of Arrb1 and miR-326 represent a feature of CSCs derived from SHH-MB. We observed that re-expression of Arrb1 and miR-326 inhibits Hh/Gli signaling pathway at multiple levels, which cause impaired proliferation and self-renewal, accompanied by down regulation of Nanog levels. In detail, miR-326 negatively regulates two components of the Hh/Gli pathway the receptor Smoothened (Smo) and the transcription factor Gli2, whereas Arrb1 suppresses the transcriptional activity of Gli1, by potentiating its p300-mediated acetylation.Our results identify a new molecular mechanism involving miR-326 and Arrb1 as regulators of SHH-MB CSCs. Specifically, low levels of Arrb1 and miR-326 trigger and maintain Hh/Gli signaling and self-renewal.
Pub.: 19 Jul '17, Pinned: 30 Sep '17
Abstract: While molecular subgrouping has revolutionized medulloblastoma classification, the extent of heterogeneity within subgroups is unknown. Similarity network fusion (SNF) applied to genome-wide DNA methylation and gene expression data across 763 primary samples identifies very homogeneous clusters of patients, supporting the presence of medulloblastoma subtypes. After integration of somatic copy-number alterations, and clinical features specific to each cluster, we identify 12 different subtypes of medulloblastoma. Integrative analysis using SNF further delineates group 3 from group 4 medulloblastoma, which is not as readily apparent through analyses of individual data types. Two clear subtypes of infants with Sonic Hedgehog medulloblastoma with disparate outcomes and biology are identified. Medulloblastoma subtypes identified through integrative clustering have important implications for stratification of future clinical trials.
Pub.: 14 Jun '17, Pinned: 30 Sep '17
Abstract: Hedgehog (Hh) signaling regulates cell fate and self-renewal in development and cancer. Canonical Hh signaling is mediated by Hh ligand binding to the receptor Patched (Ptch), which in turn activates Gli-mediated transcription through Smoothened (Smo), the molecular target of the Hh pathway inhibitors used as cancer therapeutics. Small cell lung cancer (SCLC) is a common, aggressive malignancy with universally poor prognosis. Although preclinical studies have shown that Hh inhibitors block the self-renewal capacity of SCLC cells, the lack of activating pathway mutations have cast doubt over the significance of these observations. In particular, the existence of autocrine, ligand-dependent Hh signaling in SCLC has been disputed. In a conditional Tp53;Rb1 mutant mouse model of SCLC, we now demonstrate a requirement for the Hh ligand Sonic Hedgehog (Shh) for the progression of SCLC. Conversely, we show that conditional Shh overexpression activates canonical Hh signaling in SCLC cells, and markedly accelerates tumor progression. When compared to mouse SCLC tumors expressing an activating, ligand-independent Smo mutant, tumors overexpressing Shh exhibited marked chromosomal instability and Smoothened-independent upregulation of Cyclin B1, a putative non-canonical arm of the Hh pathway. In turn, we show that overexpression of Cyclin B1 induces chromosomal instability in mouse embryonic fibroblasts lacking both Tp53 and Rb1. These results provide strong support for an autocrine, ligand-dependent model of Hh signaling in SCLC pathogenesis, and reveal a novel role for non-canonical Hh signaling through the induction of chromosomal instability.Oncogene advance online publication, 5 June 2017; doi:10.1038/onc.2017.173.
Pub.: 06 Jun '17, Pinned: 30 Sep '17
Abstract: Novel treatment options, including targeted therapies, are needed for patients with medulloblastoma (MB), especially for those with high-risk or recurrent/relapsed disease. Four major molecular subgroups of MB have been identified, one of which is characterized by activation of the sonic hedgehog (SHH) pathway. Preclinical data suggest that inhibitors of the hedgehog (Hh) pathway could become valuable treatment options for patients with this subgroup of MB. Indeed, agents targeting the positive regulator of the pathway, smoothened (SMO), have demonstrated efficacy in a subset of patients with SHH MB. However, because of resistance and the presence of mutations downstream of SMO, not all patients with SHH MB respond to SMO inhibitors. The development of agents that target these resistance mechanisms and the potential for their combination with traditional chemotherapy and SHH inhibitors will be discussed. Due to its extensive molecular heterogeneity, the future of MB treatment is in personalized therapy, which may lead to improved efficacy and reduced toxicity. This will include the development of clinically available tests that can efficiently discern the SHH subgroup. The preliminary use of these tests in clinical trials is also discussed herein.
Pub.: 22 Jun '14, Pinned: 30 Sep '17
Abstract: Aberrant Hedgehog/GLI signaling has been implicated in a diverse spectrum of human cancers, but its role in lung adenocarcinoma (LAC) is still under debate. We show that the downstream effector of the Hedgehog pathway, GLI1, is expressed in 76% of LACs, but in roughly half of these tumors, the canonical pathway activator, Smoothened, is expressed at low levels, possibly owing to epigenetic silencing. In LAC cells including the cancer stem cell compartment, we show that GLI1 is activated noncanonically by MAPK/ERK signaling. Different mechanisms can trigger the MAPK/ERK/GLI1 cascade including KRAS mutation and stimulation of NRP2 by VEGF produced by the cancer cells themselves in an autocrine loop or by stromal cells as paracrine cross talk. Suppression of GLI1, by silencing or drug-mediated, inhibits LAC cells proliferation, attenuates their stemness and increases their susceptibility to apoptosis in vitro and in vivo. These findings provide insight into the growth of LACs and point to GLI1 as a downstream effector for oncogenic pathways. Thus, strategies involving direct inhibition of GLI1 may be useful in the treatment of LACs.Oncogene advance online publication, 3 April 2017; doi:10.1038/onc.2017.91.
Pub.: 04 Apr '17, Pinned: 30 Sep '17
Abstract: Medulloblastoma (MB) arising from the cerebellum is the most common pediatric brain malignancy, with leptomeningeal metastases often present at diagnosis and recurrence associated with poor clinical outcome. In this study, we employed mouse MB models to explore the relationship of tumor pathophysiology and dysregulated expression of the Notch pathway transcription factor ATOH1, which is present in aggressive MB subtypes driven by aberrant Sonic Hedgehog/Patched (SHH/PTCH) signaling. In experiments with conditional Atoh1 mouse mutants crossed to Ptch1(+/-) mice which develop SHH-driven MB, animals with Atoh1 transgene expression developed highly penetrant MB at a young age with extensive leptomeningeal disease and metastasis to the spinal cord and brain, resembling xenografts of human SHH MB. Metastatic tumors retained abnormal SHH signaling like tumor xenografts. Conversely, Atoh1 expression was detected consistently in recurrent and metastatic SHH MB. ChIP-seq and gene expression profiling identified candidate ATOH1 targets in tumor cells involved in development and tumorigenesis. Among these targets specific to metastatic tumors, there was an enrichment in those implicated in extracellular matrix remodeling activity, cytoskeletal network and interaction with microenvironment, indicating a shift in transcriptomic and epigenomic landscapes during metastasis. Treatment with bone morphogenetic protein (BMP) or SHH pathway inhibitors decreased tumor cell proliferation and suppressed metastatic tumor growth, respectively. Our work reveals a dynamic ATOH1-driven molecular cascade underlying MB metastasis that offers possible therapeutic opportunities.
Pub.: 12 May '17, Pinned: 30 Sep '17
Abstract: Master developmental pathways, such as Notch, Wnt, and Hedgehog, are signaling systems that control proliferation, cell death, motility, migration, and stemness. These systems are not only commonly activated in many solid tumors, where they drive or contribute to cancer initiation, but also in primary and metastatic tumor development. The reactivation of developmental pathways in cancer stroma favors the development of cancer stem cells and allows their maintenance, indicating these signaling pathways as particularly attractive targets for efficient anticancer therapies, especially in advanced primary tumors and metastatic cancers. Metastasis is the worst feature of cancer development. This feature results from a cascade of events emerging from the hijacking of epithelial-mesenchymal transition, angiogenesis, migration, and invasion by transforming cells and is associated with poor survival, drug resistance, and tumor relapse. In the present review, we summarize and discuss experimental data suggesting pivotal roles for developmental pathways in cancer development and metastasis, considering the therapeutic potential. Emerging targeted antimetastatic therapies based on Notch, Wnt, and Hedgehog pathways are also discussed.
Pub.: 14 Jun '17, Pinned: 30 Sep '17
Abstract: Sonidegib (LDE225) is a potent, selective Hedgehog (Hh) inhibitor of SMOOTHENED. This study explored the safety and pharmacokinetics (PK) of sonidegib in children with relapsed/recurrent tumors followed by a phase II trial in pediatric and adult patients with relapsed medulloblastoma (MB) to assess tumor response.Pediatric patients aged ≥1-<18 years were included according to a Bayesian design starting at 372mg/m2 of continuous once daily oral sonidegib. Tumor samples were analyzed for Hh pathway activation using a validated 5-gene Hh signature assay. In phase II, pediatric patients were treated at the recommended phase II dose (RP2D) while adults received 800mg daily.Sixteen adult (16 MB) and 60 pediatric (39 MB, 21 other) patients with an age range of 2-17 years were enrolled. The RP2D of sonidegib in pediatric patients was established at 680mg/m2 once daily. The phase II study was closed prematurely. The 5-gene Hh signature assay showed that the 4 complete responders (2 pediatric and 2 adult) and 1 partial responder (adult), all had Hh‑activated tumors, while 5 patients with activated Hh had either stable disease (n=3) or progressive disease (n=2). No patients with Hh-negative signatures (n=50) responded. The safety profile for pediatric patients was generally consistent with the one established for adult patients; however, growth plate changes were observed in pre-pubertal pediatric patients'.Sonidegib was well tolerated and the RP2D in pediatric patients was 680mg/m2 once daily. Five of the 10 MB patients with activated Hh pathway demonstrated complete or partial responses.
Pub.: 13 Jun '17, Pinned: 30 Sep '17
Abstract: Tight control of the balance between self-expanding symmetric and self-renewing asymmetric neural progenitor divisions is crucial to regulate the number of cells in the developing central nervous system. We recently demonstrated that Sonic hedgehog (Shh) signalling is required for the expansion of motor neuron progenitors by maintaining symmetric divisions. Here we show that activation of Shh/Gli signalling in dividing neuroepithelial cells controls the symmetric recruitment of PKA to the centrosomes that nucleate the mitotic spindle, maintaining symmetric proliferative divisions. Notably, Shh signalling upregulates the expression of pericentrin, which is required to dock PKA to the centrosomes, which in turn exerts a positive feedback onto Shh signalling. Thus, by controlling centrosomal protein assembly, we propose that Shh signalling overcomes the intrinsic asymmetry at the centrosome during neuroepithelial cell division, thereby promoting self-expanding symmetric divisions and the expansion of the progenitor pool.
Pub.: 27 Apr '17, Pinned: 30 Sep '17
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