Anette Szczepny

Hedgehog Signaling in the Maintenance of Cancer Stem Cells.

Cancer stem cells (CSCs) represent a rare population of cells with the capacity to self-renew and give rise to heterogeneous cell lineages within a tumour. Whilst the mechanisms underlying the regulation of CSCs are poorly defined, key developmental signaling pathways required for normal stem and progenitor functions have been strongly implicated. Hedgehog (Hh) signaling is an evolutionarily-conserved pathway essential for self-renewal and cell fate determination. Aberrant Hh signaling is associated with the development and progression of various types of cancer and is implicated in multiple aspects of tumourigenesis, including the maintenance of CSCs. Here, we discuss the mounting evidence suggestive of Hh-driven CSCs in the context of haematological malignancies and solid tumours and the novel strategies that hold the potential to block many aspects of the transformation attributed to the CSC phenotype, including chemotherapeutic resistance, relapse and metastasis.

Dynamic Hedgehog signalling pathway activity in germline stem cells

Although the contribution of Hedgehog (Hh) signalling to stem cell development and oncogenesis is well recognised, its importance for spermatogonial stem cells (SSCs) has not been established. Here we interrogate adult rat SSCs using an established model in which only undifferentiated spermatogonial cells remain in the testis at 15 weeks following irradiation, and spermatogonial differentiation is induced within 4 weeks by gonadotrophin‐releasing hormone antagonist (GnRH‐ant) administration. Synthesis of Hh pathway components in untreated adult rat testes was compared with that in irradiated testes prior to and after GnRH‐ant exposure using in situ hybridization. In adult testes with complete spermatogenesis, the Desert Hedgehog ligand transcript, Dhh, was detected in Sertoli cells, some spermatogonia and in spermatocytes by in situ hybridization. Spermatogenic cells were identified as sites of Hh signalling through detection of transcripts encoding the Hh receptor, Ptc2 transcripts and proteins for the key downstream target of Hh signalling, Gli1 and the Hh transcriptional activator, Gli2. Remarkably, the undifferentiated spermatogonia present in irradiated adult rat testes contained Dhh in addition to Ptc2, Gli1 and Gli2, revealing the potential for an autocrine Hh signalling loop to sustain undifferentiated spermatogonial cells. These transcripts became undetectable by in situ hybridization following GnRH‐ant induction of spermatogonial differentiation, however, detection of Gli1 protein in spermatogonia in all groups indicates that Hh signalling is sustained. This is the first evidence of active Hh signalling in mammalian male germline stem cells, as has been documented for some cancer stem cells.

Blockade of the IL-6 trans-signalling/STAT3 axis suppresses cachexia in Kras-induced lung adenocarcinoma

Lung cancer is the leading cause of cancer death worldwide, and is frequently associated with the devastating paraneoplastic syndrome of cachexia. The potent immunomodulatory cytokine interleukin (IL)-6 has been linked with the development of lung cancer as well as cachexia; however, the mechanisms by which IL-6 promotes muscle wasting in lung cancer cachexia are ill-defined. In this study, we report that the gp130F/F knock-in mouse model displaying hyperactivation of the latent transcription factor STAT3 via the common IL-6 cytokine family signalling receptor, gp130, develops cachexia during Kras-driven lung carcinogenesis. Specifically, exacerbated weight loss, early mortality and reduced muscle and adipose tissue mass were features of the gp130F/F:KrasG12D model, but not parental KrasG12D mice in which STAT3 was not hyperactivated. Gene expression profiling of muscle tissue in cachectic gp130F/F:KrasG12D mice revealed the upregulation of IL-6 and STAT3-target genes compared with KrasG12D muscle tissue. These cachectic features of gp130F/F:KrasG12D mice were abrogated upon the genetic normalization of STAT3 activation or ablation of IL-6 in gp130F/F:KrasG12D:Stat3−/+ or gp130F/F:KrasG12D:Il6−/− mice, respectively. Furthermore, protein levels of the soluble IL-6 receptor (sIL-6R), which is the central facilitator of IL-6 trans-signalling, were elevated in cachectic muscle from gp130F/F:KrasG12D mice, and the specific blockade of IL-6 trans-signalling, but not classical signalling, with an anti-IL-6R antibody ameliorated cachexia-related characteristics in gp130F/F:KrasG12D mice. Collectively, these preclinical findings identify trans-signalling via STAT3 as the signalling modality by which IL-6 promotes muscle wasting in lung cancer cachexia, and therefore support the clinical evaluation of the IL-6 trans-signalling/STAT3 axis as a therapeutic target in advanced lung cancer patients presenting with cachexia.

The role of canonical and non-canonical Hedgehog signaling in tumor progression in a mouse model of small cell lung cancer

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.

Overlapping binding sites for importin β1 and suppressor of fused (SuFu) on glioma-associated oncogene homologue 1 (Gli1) regulate its nuclear localization

A key factor in oncogenesis is the transport into the nucleus of oncogenic signalling molecules, such as Gli1 (glioma-associated oncogene homologue 1), the central transcriptional activator in the Hedgehog signalling pathway. Little is known, however, how factors such as Gli are transported into the nucleus and how this may be regulated by interaction with other cellular factors, such as the negative regulator suppressor of fused (SuFu). In the present study we show for the first time that nuclear entry of Gli1 is regulated by a unique mechanism through mutually exclusive binding by its nuclear import factor Impβ1 (importin β1) and SuFu. Using quantitative live mammalian cell imaging, we show that nuclear accumulation of GFP-Gli1 fusion proteins, but not of a control protein, is specifically inhibited by co-expression of SuFu. Using a direct binding assay, we show that Impβ1 exhibits a high nanomolar affinity to Gli1, with specific knockdown of Impβ1 expression being able to inhibit Gli1 nuclear accumulation, thus implicating Impβ1 as the nuclear transporter for Gli1 for the first time. SuFu also binds to Gli1 with a high nanomolar affinity, intriguingly being able to compete with Impβ1 for binding to Gli1, through the fact that the sites for SuFu and Impβ1 binding overlap at the Gli1 N-terminus. The results indicate for the first time that the relative intracellular concentrations of SuFu and Impβ1 are likely to determine the localization of Gli1, with implications for its action in cancer, as well as in developmental systems.

The tumor suppressor Hic1 maintains chromosomal stability independent of Tp53

Hypermethylated-in-Cancer 1 (Hic1) is a tumor suppressor gene frequently inactivated by epigenetic silencing and loss-of-heterozygosity in a broad range of cancers. Loss of HIC1, a sequence-specific zinc finger transcriptional repressor, results in deregulation of genes that promote a malignant phenotype in a lineage-specific manner. In particular, upregulation of the HIC1 target gene SIRT1, a histone deacetylase, can promote tumor growth by inactivating TP53. An alternate line of evidence suggests that HIC1 can promote the repair of DNA double strand breaks through an interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex. Using a conditional knockout mouse model of tumor initiation, we now show that inactivation of Hic1 results in cell cycle arrest, premature senescence, chromosomal instability and spontaneous transformation in vitro. This phenocopies the effects of deleting Brca1, a component of the homologous recombination DNA repair pathway, in mouse embryonic fibroblasts. These effects did not appear to be mediated by deregulation of Hic1 target gene expression or loss of Tp53 function, and rather support a role for Hic1 in maintaining genome integrity during sustained replicative stress. Loss of Hic1 function also cooperated with activation of oncogenic KRas in the adult airway epithelium of mice, resulting in the formation of highly pleomorphic adenocarcinomas with a micropapillary phenotype in vivo. These results suggest that loss of Hic1 expression in the early stages of tumor formation may contribute to malignant transformation through the acquisition of chromosomal instability.

Inhibition of activin signaling in lung adenocarcinoma increases the therapeutic index of platinum chemotherapy

Inhibition of activin signaling enhances the efficacy and safety of platinum chemotherapy in lung adenocarcinoma models. Blocking activin actively treats cancer Platinum-based chemotherapy is a mainstay of treatment for lung cancer, but resistance to this therapy is a common problem, as are dose-limiting side effects, particularly kidney toxicity. To search for mechanisms that may contribute to treatment resistance, Marini et al. performed a whole-genome RNA interference screen and identified the activin pathway, which can be targeted. The authors demonstrated that inhibition of this pathway using a small molecule or a protein called follistatin can offer a dual benefit in that it potentiates the effects of platinum drugs in mouse models of cancer and also protects the animals from kidney damage. These findings suggest that activin inhibitors could be a valuable addition to platinum chemotherapy, enhancing the efficacy of treatment while also allowing the use of higher doses or longer periods of drug exposure. Resistance to platinum chemotherapy is a long-standing problem in the management of lung adenocarcinoma. Using a whole-genome synthetic lethal RNA interference screen, we identified activin signaling as a critical mediator of innate platinum resistance. The transforming growth factor–β (TGFβ) superfamily ligands activin A and growth differentiation factor 11 (GDF11) mediated resistance via their cognate receptors through TGFβ-activated kinase 1 (TAK1), rather than through the SMAD family of transcription factors. Inhibition of activin receptor signaling or blockade of activin A and GDF11 by the endogenous protein follistatin overcame this resistance. Consistent with the role of activin signaling in acute renal injury, both therapeutic interventions attenuated acute cisplatin-induced nephrotoxicity, its major dose-limiting side effect. This cancer-specific enhancement of platinum-induced cell death has the potential to dramatically improve the safety and efficacy of chemotherapy in lung cancer patients.

Deep multi-region whole-genome sequencing reveals heterogeneity and gene-by-environment interactions in treatment-naive, metastatic lung cancer

Our understanding of genomic heterogeneity in lung cancer is largely based on the analysis of early-stage surgical specimens. Here we used endoscopic sampling of paired primary and intrathoracic metastatic tumors from 11 lung cancer patients to map genomic heterogeneity inoperable lung cancer with deep whole-genome sequencing. Intra-patient heterogeneity in driver or targetable mutations was predominantly in the form of copy number gain. Private mutation signatures, including patterns consistent with defects in homologous recombination, were highly variable both within and between patients. Irrespective of histotype, we observed a smaller than expected number of private mutations, suggesting that ancestral clones accumulated large mutation burdens immediately prior to metastasis. Single-region whole-genome sequencing of from 20 patients showed that tumors in ever-smokers with the strongest tobacco signatures were associated with germline variants in genes implicated in the repair of cigarette-induced DNA damage. Our results suggest that lung cancer precursors in ever-smokers accumulate large numbers of mutations prior to the formation of frank malignancy followed by rapid metastatic spread. In advanced lung cancer, germline variants in DNA repair genes may interact with the airway environment to influence the pattern of founder mutations, whereas similar interactions with the tumor microenvironment may play a role in the acquisition of mutations following metastasis.

Abstract 3360: Targeted catalytic inhibition of EZH2 synergizes with low-dose HDACi in malignant rhabdoid tumors

Malignant Rhabdoid Tumor (MRT) is a rare pediatric cancer of the kidney and CNS that is resistant to current treatment protocols. MRT is genetically characterized by homozygous inactivation of SMARCB1, a critical subunit of the SWI/SNF chromatin-remodeling complex. Next-generation sequencing data suggests that inactivation of SMARCB1 is the primary driver mutation, implicating epigenetic deregulation in the pathogenesis of MRT. Recently, we showed that sustained treatment of MRT cell lines with low-dose Panobinostat (LBH589), inhibited tumor growth by driving multi-lineage differentiation in vitro and in vivo. Furthermore, re-expression of physiological levels of SMARCB1 in G401 MRT cells phenocopied the low-dose LBH589 treatment and led to growth inhibition, senescence and terminal differentiation in vitro and in vivo. Enhancer of Zeste homolog 2 (EZH2), a core subunit of the Polycomb Repressive Complex 2 (PRC2), confers transcriptional silencing via the addition of methyl groups to Lysine 27 of Histone 3 (H3K27me 3 ), and is a transcriptional target of SMARCB1. EZH2 expression and H3K27me 3 were drastically reduced following sustained low-dose LBH589 treatment and re-expression of SMARCB1 in G401 MRT cells. Sustained siRNA knockdown of EZH2 in G401 cells resulted in reduced cell growth and changes in mRNA expression similar to those observed following low-dose LBH589 treatment and SMARCB1 re-expression. Treatment of MRT cells with the EZH2-catalytic domain inhibitor, GSK-126, had no effect on EZH2 expression and only partially reduced H3K27me 3 and cell growth at doses 1nM-10μM suggesting important non-catalytic EZH2 function. However, MRT cells treated in combination with low-dose LBH589 and GSK-126, lost EZH2 and H3K27me 3 expression and exhibited significantly reduced cell growth in vitro compared to single agent controls, revealing a synergistic relationship. Similar effects were observed in an in vivo xenograft model, with low-dose LBH589 and GSK-126 treatment leading to a marked reduction in tumor growth, not observed with single agent treatment. This data suggests EZH2 is an important mediator of MRT proliferation and differentiation and provides evidence for dual therapeutic targeting of EZH2 with low-dose HDACi in MRT. Citation Format: Dean Popovski, Elizabeth M. Algar, Catherine R. Cochrane, Anette Szczepny, W. Samantha Jayasekara, David M. Ashley, Peter Downie, D. Neil Watkins, Jason E. Cain. Targeted catalytic inhibition of EZH2 synergizes with low-dose HDACi in malignant rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3360. doi:10.1158/1538-7445.AM2017-3360

276. Targets of the action of nuclear transport factors in spermatogenesis

During spermatogenesis, precise and orderly switches in gene expression are required. The movement of transcription factors (TFs) and nuclear proteins into and out of the nucleus is highly regulated, thus determining the extent and timing of gene expression. Most nuclear transport events are mediated by members of the importin superfamily that specifically recognise their cargoes and facilitate the passage of receptor-substrate complexes through the nuclear pore complex (NPC) which is made up of nucleoporin proteins. Eight human importin α isoforms are known that function in heterodimeric form with importin β whilst there are 20 members of the importin β family, which mediate the nuclear import or export of a very diverse set of protein or RNA cargoes. Understanding of importin and TF/chromatin component interaction during spermatogenesis should identify potential developmental switches, critical steps in the spermatogenic process. We are interested in the expression of different importins during spermatogenesis, and their specific nuclear import/export substrates as candidates in developmental switches. Preliminary analysis has shown that the nuclear import factors importin β1 and 3 are not only expressed in germ cells, but also alter their cellular distribution during maturation. In a yeast two-hybrid screen, using truncated importin β3 as bait and a library made from adult mouse testis, we identified a transcriptional repressor gene involved in cell cycle regulation, and an enzyme of the purine nucleotide biosynthesis pathway as candidate binding partners. Further studies will focus on elucidating the biological significance of these interactions in spermatogenesis.