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Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by loss of function TP53 and RB1 mutations in addition to mutations in other oncogenes including MYC. Overexpression of MYC together with Trp53 and Rb1 loss in pulmonary neuroendocrine cells of the mouse lung drives an aggressive neuroendocrine low variant subtype of SCLC. However, the transforming potential of MYC amplification alone on airway epithelium is unclear. Therefore, we selectively and conditionally overexpressed MYC stochastically throughout the airway or specifically in neuroendocrine, club, or alveolar type II cells in the adult mouse lung. We observed that MYC overexpression induced carcinoma in situ which did not progress to invasive disease. The formation of adenoma or SCLC carcinoma in situ was dependent on the cell of origin. In contrast, MYC overexpression combined with conditional deletion of both Trp53 and Rb1 exclusively gave rise to SCLC, irrespective of the cell lineage of origin. However, cell of origin influenced disease latency, metastatic potential, and the transcriptional profile of the SCLC phenotype. Together this reveals that MYC overexpression alone provides a proliferative advantage but when combined with deletion of Trp53 and Rb1 it facilitates the formation of aggressive SCLC from multiple cell lineages.

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.

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.

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.

Spermatogenic differentiation requires progressive gene expression changes, and proteins required for this must be transported into the nucleus. Many of these contain a nuclear localization signal and are likely to be transported by importin protein family members, each of which recognizes and transports distinct cargo proteins. We hypothesized that importins, as modulators of protein nuclear access, would display distinct expression profiles during spermatogenesis, indicating their potential to regulate key steps in cellular differentiation. This was tested throughout testicular development in rodents. Real-time PCR analysis of postnatal mouse testes revealed changing expression levels of Knpb1 (encoding importin beta 1) and Ranbp5 (encoding beta 3) mRNAs, with Knpb1 highest at 26 days postpartum and Ranbp5 highest in Day 26 and adult testis. Their distinctive cellular expression patterns visualized using in situ hybridization and immunohistochemistry were identical in mouse and rat testes where examined. Within the seminiferous epithelium, Knpb1 mRNA and importin beta1 protein were detected within mitotic Sertoli and germ cells during fetal and early postnatal development, becoming restricted to spermatogonia and spermatocytes in adulthood. Importin beta 3 protein in fetal germ cells displayed a striking difference in intracellular localization between male and female gonads. In adult testes, Ranbp5 mRNA was detected in round spermatids and importin beta 3 protein in elongating spermatids. This is the first comprehensive in situ demonstration of developmentally regulated synthesis of nuclear transport components. The contrasting expression patterns of importins beta 1 and 3 identify them as candidates for regulating nuclear access of factors required for developmental switches.

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.

HIC1 is a zinc-finger transcriptional repressor frequently targeted for deletion and/or epigenetic gene silencing in cancer. In cancer cell lines, re-expression of HIC1 results in the downregulation of target genes thought to promote context-dependent tumor growth, including Sirt1, Atoh1, Efna1 and components of the WNT and STAT3 signalling pathway. In mouse embryonic fibroblasts, we now show that deletion of a conditional loxP allele of Hic1 results in immediate growth arrest without significant overexpression of Hic1 target genes, followed by a prolonged period of cellular quiescence. After several weeks, we then observed the spontaneous emergence of rapidly growing clones that fulfil all the criteria for transformation, associated with aneuploidy and recurrent copy number alterations. By contrast, conditional deletion of p53 using the same experimental protocol resulted in prompt immortalisation without transformation, and a stable tetraploid karyotype resembling that seen in NIH-3T3 cells. These data suggest that inactivation of Hic1 in cells undergoing replication stress in tissue culture can induce complete transformation associated with marked genomic instability. To test this hypothesis in vivo, we employed the inhaled adenoviral Cre recombinase model in order to direct loxP recombination to the airway epithelium of adult mice. As previously described by other groups, Cre activation of a conditional oncogenic KRasG12D mutant resulted in the appearance of multiple lung adenomas within 6-8 weeks following viral inhalation. By contrast, mice carrying this KRasG12D allele, as well as homozygous for the conditional Hic1lox knockout allele, developed highly aggressive adenocarcinomas with prominent pleomorphic and micropapillary histology. These data suggest a novel tumour suppressor role for HIC1 in maintaining genomic stability in replicating cells. HIC1 is known to interact with components of the SWI/SNF and PRC2 complexes, which help maintain genomic integrity by re-establishing constitutive heterochromatin following DNA replication. We therefore speculate that loss of HIC1 function may phenocopy recently described mutations in components of these chromatin-remodelling complexes such as ARID1A and BRG1. Citation Format: Anette Szczepny, Lisa McKenzie, Samantha Jayesekara, Prue Russell, Gavin Wright, Stephen B. Baylin, Fernando J. Rossello, Jason E. Cain, David N. Watkins. Conditional deletion of the tumor suppressor Hic1 results in aneuploidy and single-step transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-81. doi:10.1158/1538-7445.AM2014-LB-81

Hedgehog (Hh) signalling regulates normal development of many tissues and is upregulated in some cancers. Mice which lack the testicular desert hedgehog (Dhh) ligand exhibit disrupted embryonic gonad formation and male infertility in adulthood. However, the roles and sites of Hedgehog (Hh) signalling activity in the developing rat testis are unknown. Transcripts encoding Hh pathway components in embryonic and juvenile rat testes were localised by in situ hybridization with DIG-labelled cRNA probes. On embryonic day (E) 17.5, Sertoli cells contained transcripts encoding Dhh and both gonocytes and Sertoli cells contained the Ptc2 and Smo receptor transcripts. The cytoplasmic regulators (fused [Fu], suppressor of fused [SuFu]) and the three Gli transcriptional mediators were also detected in gonocytes. On E21.5 and postnatal day 4, all transcripts were present in Sertoli cells, but not gonocytes. To test the function of Hh signalling at the onset of rodent spermatogenesis, the newborn (day 1) mouse testis was cultured in hanging drop cultures. Addition of the selective Hh signalling inhibitor, cyclopamine, yielded a significantly lower level of the Hh target, Gli1 mRNA, as measured by real time PCR compared to vehicle controls. This confirms Hh pathway activity and inhibition in this system. Meiotic markers, SYCP3 and Stra8, were consistently upregulated (n = 6) following cyclopamine addition, however the fold-change varied between experiments. Overall, the cellular expression data indicate that Hh signals are active in both embryonic and juvenile rodent testes. The finding that Hh genes analysed in this study are expressed in both Sertoli and germ cells shows that both cell types are potential Hh targets, depending on the developmental stage of testis. The upregulation of candidates for Dhh target genes in the juvenile mouse testis in vitro suggests that Hedgehog signaling downregulates the expression of meiotic genes in gonocytes via paracrine mechanisms.

In normal tissues, Hedgehog-induced progenitor cell proliferation is transient and tightly regulated, preventing continuous regeneration. However, activation of constitutive Hedgehog signalling results in unregulated self-renewal of progenitor cells in association with several human cancers. Although the contribution of Hedgehog signalling to cancers is widely accepted, its impact on spermatogonial stem cells and impact on male fertility are unknown. In this study, we aimed to clarify the possible role of Hh signalling on normal spermatogenesis in the adult rat and in adult testicular stem cells in the irradiated model {1}. Adult male rats were obtained from Monash University Central Animal Service and killed by cervical dislocation before tissue removal and fixation in Bouins for routine histochemical procedures. For studies on irradiated testes, adult LBNF1 male rats (hybrids between Lewis and Brown–Norway) were purchased from Harlan Sprague–Dawley, Inc. (Indianapolis, IN, USA). Testes were irradiated with 6 Gy to deplete all maturing germ cell types. At 15 weeks after irradiation the animals were injected simultaneously with 1.5 mg each of Cetrorelix pamoate and Cetrorelix acetate. Testes were collected 1, 2 or 4 weeks after injection. In situ hybridisation combined with immunohistochemistry was performed using DIG-labelled cRNA probes to identify the cells in which Hedgehog signalling components are made {2}. Signals for mRNAs encoding t he transmembrane receptors Ptc2 and Smo are most intensely detected in spermatogonia and spermatocytes and are much less intense in the round spermatids. The mRNA for the cytoplamic regulator, Fused, is restricted to the earliest germ cell types, whereas expression of the negative cytoplasmic regulator, SuFu, only begins in the round spermatids and persists in elongating spermatids. Gli1 and Gli3 are expressed from spermatogonia through to round spermatids, whereas Gli2 is restricted to spermatogonia and spermatocytes. This pattern mimics what was reported for mouse {2}. Examination of the irradiated rat testis model revealed that Hedgehog signalling machinery is produced by resting spermatogonial stem cells but is turned off when they differentiate in response to hormones. This matches the emerging understanding of Hedgehog signals in cancer stem cells and provides the first demonstration that Hedgehog signalling may influence stem cells in the adult testis. (1) Shuttlesworth G.A. et al. 2000. Endocrinology. 141: 37–49 (2) Szczepny A. et al. 2006. Dev Dyn. 235:3063–3070.

Hedgehog (Hh) signalling is best known for its involvement in regulating patterning, driving cell proliferation, promoting cell survival and directing differentiation during embryonic development.1 The role that Hedgehog signalling plays in the testis is not yet clearly defined, though deletion of one Hedgehog ligand, Dhh, leads to male infertility. The Gli family of zinc finger TFs, consisting of Gli1, Gli2 and Gli3, are mediators of the Hh signalling cascade in vertebrates. We have previously shown that the mRNA transcripts encoding all three Glis in the adult mouse testis are expressed highly in spermatogonia, spermatocytes and to a lower extent in the round spermatids. To understand the potential sites of action of Hh proteins in spermatogenesis, we have extended our analysis to other genes involved in the Hh signalling pathway in the adult mouse testis. Using in situ hybridization, Patched2, a transmembrane receptor for Hh, was detected in spermatogonia and spermatocytes, with an apparently lower expression in the round spermatids. The mRNA of Smoothened, another transmembrane protein which forms a membrane receptor complex with Patched, is highly expressed in spermatogonia and spermatocytes, again showing lower expression in round spermatids and interstitial cells. Fused, a positive regulator of Hh signalling, is highly expressed in spermatogonia and spermatocytes with slightly lower expression in round spermatids. SuFu is a negative regulator of Hh signalling, known to repress Gli1 function in part by tethering it in the cytoplasm. The mRNA encoding SuFu is absent from spermatogonia, detected in spermatocytes and persists in round spermatids where its expression appears highest, suggesting that the SuFu protein may be acting to switch off Hh signalling at that stage of spermatogenesis. Overall, the regulated expression pattern of these genes in the adult mouse testis suggests a role for Hh signalling in the regulation of spermatogenesis. (1)Ruiz i Altaba A. (1999) Trends Genet. 15(10), 418–425.

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.

Development is highly regulated by complex signalling cascades. One such pathway is the Hedgehog (Hh) signalling pathway which plays an essential role in spermatogenesis. The Gli family of zinc finger TFs, consisting of Gli1, Gli2 and Gli3, are mediators of the Hh signalling cascade. Gli1 is an activator of Hh target genes, whereas Gli2 and Gli3 can undergo proteolytic cleavage and function as both activators and repressors. Little is known regarding the nuclear import pathway of these TFs. In this study, the mRNA expression pattern of all Gli family members in the developing mouse testis was compiled by in situ hybridisation and shown to have unique expression patterns. In the adult mouse testis, Gli1 mRNA was detected in spermatogonia through to round spermatids whereas Gli2 was only found in spermatogonia and spermatocytes. Very low levels of Gli3 mRNA were detected in all ages and cell types. Since little is known regarding the import pathway for Gli1, expression vectors containing different fragments of the N-terminus of Gli1 were created and used to perform transfection experiments and generate vectors for bacterial GFP-fusion protein expression. Transfection experiments into African green monkey kidney Cos-7 cells, and the murine spermatogenic cell lines, Gc-1 and Gc-2 using 3 different constructs localised the NLS(s) required to target Gli1 to the nucleus in the zinc finger DNA-binding domain of Gli1. Preliminary results for in vitro binding of bacterially expressed Gli1 indicated no binding by importin β 1 or β3 but a weak interaction with the importin α/β heterodimer. This can be seen as the first step towards defining the nuclear import pathway for Gli1. The mechanisms by which Gli activity is modulated remain unanswered and the regulation of its nuclear entry may be an important means of doing so.

Spermatogenesis requires progression of a self-renewing male germline stem cell population through a precisely timed and ordered developmental sequence to form spermatozoa. For several years, we have been investigating the functional impact of signals on this progression by members of the TGFβ superfamily, follicle stimulating hormone and the bcl-2 family. For example, our lab and others have shown that activin A, bone morphogenetic protein (BMP)-4 and glial-derived neurotropic factor (GDNF) all modulate stem cell development and spermatogonial differentiation at the onset of spermatogenesis after birth. We are trying to understand how germ cells 'interpret' this plethora of competing signals to mediate maturation. Progression through successive maturation states in response to such signals requires the movement of proteins, including transcription factors, into the nucleus to implement changes in gene transcription. We are investigating the concept that regulated transport of proteins into the nucleus is one mechanism that governs spermatogenic differentiation, and we have focused on analysis of the major class of nuclear transport factors, the importins (IMPs). A diverse family comprising at least six different α forms and 20 different β forms in the mouse, the IMP proteins selectively bind a diverse range of cargo proteins and mediate their passage through the nuclear pore complex and into the nucleus. Our immunohistochemical and in situ hybridisation studies have demonstrated developmentally regulated expression of many IMPs in germ cells, in the fetus, in the neonate and in the adult. This suggests that they function to transport cargo required for discrete stages of spermatogenesis. Our recent studies examined the mouse embryonic gonad around the time when specification to form either a testis or an ovary occurs. The IMPβ3 protein is present in both male and female germ cells, but the subcellular localisation and expression patterns within these cell types is gender- and age-specific. We are currently exploring the functional significance of our observations.

We present a detailed study of the expression pattern of WD repeat and SOCS box-containing 2 (Wsb2) in mouse embryonic and adult gonads. Wsb2 was previously identified in a differential screen aimed at identifying the genes involved in male- and female-specific gonadal development. Wsb2 expression was analysed during mouse gonadogenesis by real-time PCR, whole-mount and section in situ hybridisation and immunofluorescence. Wsb2 mRNA expression was initially detected in gonads of both sexes from 11.5 days post coitum (dpc) until 12.0 dpc. By 12.5 dpc and thereafter, Wsb2 expression rapidly decreased in the female, while persisting in the male gonads. In foetal, newborn and juvenile testes, Wsb2 mRNA and protein were readily detected in the seminiferous cords within both Sertoli and germ cells. Wsb2 mRNA was present in spermatogonia, spermatocytes and in Sertoli cells of the adult mouse testis. The differential expression of Wsb2 in male versus female embryonic gonads suggests some male-specific role in gonad development, and its expression in the first wave of spermatogenesis indicates a role in germ cells. Real-time analysis of adult mouse testis tubules cultured in the presence of the Hedgehog signalling inhibitor, cyclopamine, showed a downregulation of Wsb2 mRNA after treatment which suggests that Wsb2 may be a target of Hedgehog signalling.