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.

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.

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.

Expression of Wsb2 in the developing and adult mouse testis.

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.