Simon Junankar

Tumor-induced anorexia and weight loss are mediated by the TGF-beta superfamily cytokine MIC-1.

Anorexia and weight loss are part of the wasting syndrome of late-stage cancer, are a major cause of morbidity and mortality in cancer, and are thought to be cytokine mediated. Macrophage inhibitory cytokine-1 (MIC-1) is produced by many cancers. Examination of sera from individuals with advanced prostate cancer showed a direct relationship between MIC-1 abundance and cancer-associated weight loss. In mice with xenografted prostate tumors, elevated MIC-1 levels were also associated with marked weight, fat and lean tissue loss that was mediated by decreased food intake and was reversed by administration of antibody to MIC-1. Additionally, normal mice given systemic MIC-1 and transgenic mice overexpressing MIC-1 showed hypophagia and reduced body weight. MIC-1 mediates its effects by central mechanisms that implicate the hypothalamic transforming growth factor-β receptor II, extracellular signal–regulated kinases 1 and 2, signal transducer and activator of transcription-3, neuropeptide Y and pro-opiomelanocortin. Thus, MIC-1 is a newly defined central regulator of appetite and a potential target for the treatment of both cancer anorexia and weight loss, as well as of obesity.

The Propeptide Mediates Formation of Stromal Stores of PROMIC-1: Role in Determining Prostate Cancer Outcome

The extracellular matrix (ECM) is a reservoir of cellular binding proteins and growth factors that are critical for normal cell behavior, and aberrations in the ECM invariably accompany malignancies such as prostate cancer. Carcinomas commonly overexpress macrophage inhibitory cytokine 1 (MIC-1), a proapoptotic and antitumorigenic transforming growth factor-beta superfamily cytokine. Here we show that MIC-1 is often secreted in an unprocessed propeptide containing form. It is variably processed intracellularly, with unprocessed forms being secreted from several tumor lines, including prostate carcinoma lines, PC-3 and LNCaP. Once secreted, only unprocessed proMIC-1 binds ECM, demonstrating for the first time the occurrence of extracellular stores of MIC-1. The propeptide mediates this association via its COOH-terminal 89 amino acids. Xenograft models bearing tumors secreting various engineered forms of MIC-1 show that the propeptide regulates the balance between ECM stores and circulating serum levels of mature MIC-1 in vivo. The absence of propeptide results in approximately 20-fold increase in serum MIC-1 levels. The significance of stromal MIC-1 stores was evaluated in prostate cancer tissue cores, which show major variation in stromal levels of MIC-1. Stromal MIC-1 levels are linked to prostate cancer outcome following radical prostatectomy, with decreasing stromal levels providing an important independent predictor of disease relapse. In low-grade localized prostate cancer (Gleason sum score < or = 6), the level of MIC-1 stromal stores was the best predictor of future relapse when compared with all other clinicopathologic variables. The secretion and ECM association of unprocessed proMIC-1 is likely to play a central role in modulating local bioavailability of MIC-1 which can affect patient outcome in prostate cancer and other epithelial tumors.

Real-Time Intravital Imaging Establishes Tumor-Associated Macrophages as the Extraskeletal Target of Bisphosphonate Action in Cancer

UNLABELLED Recent clinical trials have shown that bisphosphonate drugs improve breast cancer patient survival independent of their antiresorptive effects on the skeleton. However, because bisphosphonates bind rapidly to bone mineral, the exact mechanisms of their antitumor action, particularly on cells outside of bone, remain unknown. Here, we used real-time intravital two-photon microscopy to show extensive leakage of fluorescent bisphosphonate from the vasculature in 4T1 mouse mammary tumors, where it initially binds to areas of small, granular microcalcifications that are engulfed by tumor-associated macrophages (TAM), but not tumor cells. Importantly, we also observed uptake of radiolabeled bisphosphonate in the primary breast tumor of a patient and showed the resected tumor to be infiltrated with TAMs and to contain similar granular microcalcifications. These data represent the first compelling in vivo evidence that bisphosphonates can target cells in tumors outside the skeleton and that their antitumor activity is likely to be mediated via TAMs. SIGNIFICANCE Bisphosphonates are assumed to act solely in bone. However, mouse models and clinical trials show that they have surprising antitumor effects outside bone. We provide unequivocal evidence that bisphosphonates target TAMs, but not tumor cells, to exert their extraskeletal effects, offering a rationale for use in patients with early disease.

c-Myc and Her2 cooperate to drive a stem-like phenotype with poor prognosis in breast cancer

The HER2 (ERBB2) and MYC genes are commonly amplified in breast cancer, yet little is known about their molecular and clinical interaction. Using a novel chimeric mammary transgenic approach and in vitro models, we demonstrate markedly increased self-renewal and tumour-propagating capability of cells transformed with Her2 and c-Myc. Coexpression of both oncoproteins in cultured cells led to the activation of a c-Myc transcriptional signature and acquisition of a self-renewing phenotype independent of an epithelial–mesenchymal transition programme or regulation of conventional cancer stem cell markers. Instead, Her2 and c-Myc cooperated to induce the expression of lipoprotein lipase, which was required for proliferation and self-renewal in vitro. HER2 and MYC were frequently coamplified in breast cancer, associated with aggressive clinical behaviour and poor outcome. Lastly, we show that in HER2+ breast cancer patients receiving adjuvant chemotherapy (but not targeted anti-Her2 therapy), MYC amplification is associated with a poor outcome. These findings demonstrate the importance of molecular and cellular context in oncogenic transformation and acquisition of a malignant stem-like phenotype and have diagnostic and therapeutic consequences for the clinical management of HER2+ breast cancer.

ID4 controls mammary stem cells and marks breast cancers with a stem cell-like phenotype

Basal-like breast cancer (BLBC) is a heterogeneous disease with poor prognosis; however, its cellular origins and aetiology are poorly understood. In this study, we show that inhibitor of differentiation 4 (ID4) is a key regulator of mammary stem cell self-renewal and marks a subset of BLBC with a putative mammary basal cell of origin. Using an ID4GFP knock-in reporter mouse and single-cell transcriptomics, we show that ID4 marks a stem cell-enriched subset of the mammary basal cell population. ID4 maintains the mammary stem cell pool by suppressing key factors required for luminal differentiation. Furthermore, ID4 is specifically expressed by a subset of human BLBC that possess a very poor prognosis and a transcriptional signature similar to a mammary stem cell. These studies identify ID4 as a mammary stem cell regulator, deconvolute the heterogeneity of BLBC and link a subset of mammary stem cells to the aetiology of BLBC.

Interleukin-27 Signaling Promotes Immunity against Endogenously Arising Murine Tumors

Interleukin-27 (IL-27) is a pleiotropic cytokine but its immunosuppressive effects predominate during many in vivo immunological challenges. Despite this, evidence from tumor cell line transfer models suggested that IL-27 could promote immune responses in the tumor context. However, the role of IL-27 in immunity against tumors that develop in situ and in tumor immunosurveillance remain undefined. In this study, we demonstrate that tumor development and growth are accelerated in IL-27 receptor α (Il27ra)-deficient mice. Enhanced tumor growth in both carcinogen-induced fibrosarcoma and oncogene-driven mammary carcinoma was associated with decreased interferon-γ production by CD4 and CD8 T cells and increased numbers of regulatory T-cells (Treg). This is the first study to show that IL-27 promotes protective immune responses against endogenous tumors, which is critical as the basis for future development of an IL-27 based therapeutic agent.

Redefining the expression and function of the inhibitor of differentiation 1 in mammary gland development

The accumulation of poorly differentiated cells is a hallmark of breast neoplasia and progression. Thus an understanding of the factors controlling mammary differentiation is critical to a proper understanding of breast tumourigenesis. The Inhibitor of Differentiation 1 (Id1) protein has well documented roles in the control of mammary epithelial differentiation and proliferation in vitro and breast cancer progression in vivo. However, it has not been determined whether Id1 expression is sufficient for the inhibition of mammary epithelial differentiation or the promotion of neoplastic transformation in vivo. We now show that Id1 is not commonly expressed by the luminal mammary epithelia, as previously reported. Generation and analysis of a transgenic mouse model of Id1 overexpression in the mammary gland reveals that Id1 is insufficient for neoplastic progression in virgin animals or to prevent terminal differentiation of the luminal epithelia during pregnancy and lactation. Together, these data demonstrate that there is no luminal cell-autonomous role for Id1 in mammary epithelial cell fate determination, ductal morphogenesis and terminal differentiation.

Could the properties of IL-27 make it an ideal adjuvant for anticancer immunotherapy?

We have recently been the first to demonstrate that interleukin (IL)-27 protects against the emergence and progression of autochthonous tumors. Accumulating evidence suggests that IL-27 might be uniquely well positioned to amplify beneficial TH1 anticancer immune responses while suppressing the unwanted accumulation of regulatory T cells.

High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes

High-throughput single-cell RNA-Sequencing is a powerful technique for gene expression profiling of complex and heterogeneous cellular populations such as the immune system. However, these methods only provide short-read sequence from one end of a cDNA template, making them poorly suited to the investigation of gene-regulatory events such as mRNA splicing, adaptive immune responses or somatic genome evolution. To address this challenge, we have developed a method that combines targeted long-read sequencing with short-read based transcriptome profiling of barcoded single cell libraries generated by droplet-based partitioning. We use Repertoire And Gene Expression sequencing (RAGE-seq) to accurately characterize full-length T cell (TCR) and B cell (BCR) receptor sequences and transcriptional profiles of more than 7,138 lymphocytes sampled from the primary tumour and draining lymph node of a breast cancer patient. With this method we show that somatic mutation, alternate splicing and clonal evolution of T and B lymphocytes can be tracked across these tissue compartments. Our results demonstrate that RAGE-Seq is an accessible and cost-effective method for high-throughput deep single cell profiling, applicable to a wide range of biological challenges.

A novel role for the HLH protein Inhibitor of Differentiation 4 (ID4) in the DNA damage response in basal-like breast cancer.

Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of Differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC, through unknown mechanisms. Here, we have defined a molecular mechanism of action for ID4 in BLBC and the related disease highgrade serous ovarian cancer (HGSOV), by combining RIME proteomic analysis and ChIP-Seq mapping of genomic binding sites. Remarkably, these studies have revealed novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage and regulating DNA damage signalling. Clinical analysis demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair pathways. These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOV.