Combination of bazedoxifene with chemotherapy and SMAC-mimetics for the treatment of colorectal cancer.

Excessive STAT3 signalling via gp130, the shared receptor subunit for IL-6 and IL-11, contributes to disease progression and poor survival outcomes in patients with colorectal cancer. Here, we provide evidence that bazedoxifene inhibits tumour growth via direct interaction with the gp130 receptor to suppress IL-6 and IL-11-mediated STAT3 signalling. Additionally, bazedoxifene combined with chemotherapy synergistically reduced cell proliferation and induced apoptosis in patient-derived colon cancer organoids. We elucidated that the primary mechanism of anti-tumour activity conferred by bazedoxifene treatment occurs via pro-apoptotic responses in tumour cells. Co-treatment with bazedoxifene and the SMAC-mimetics, LCL161 or Birinapant, that target the IAP family of proteins, demonstrated increased apoptosis and reduced proliferation in colorectal cancer cells. Our findings provide evidence that bazedoxifene treatment could be combined with SMAC-mimetics and chemotherapy to enhance tumour cell apoptosis in colorectal cancer, where gp130 receptor signalling promotes tumour growth and progression.

Mechanisms of cellular crosstalk in the gastric tumor microenvironment are mediated by YAP1 and STAT3.

Deregulation of the Hippo pathway is a driver for cancer progression and treatment resistance. In the context of gastric cancer, YAP1 is a biomarker for poor patient prognosis. Although genomic tumor profiling provides information of Hippo pathway activation, the present study demonstrates that inhibition of Yap1 activity has anti-tumor effects in gastric tumors driven by oncogenic mutations and inflammatory cytokines. We show that Yap1 is a key regulator of cell metabolism, proliferation, and immune responses in normal and neoplastic gastric epithelium. We propose that the Hippo pathway is targetable across gastric cancer subtypes and its therapeutic benefits are likely to be mediated by both cancer cell-intrinsic and -extrinsic mechanisms.

Therapeutic inhibition of the SRC-kinase HCK facilitates T cell tumor infiltration and improves response to immunotherapy.

Although immunotherapy has revolutionized cancer treatment, many immunogenic tumors remain refractory to treatment. This can be largely attributed to an immunologically "cold" tumor microenvironment characterized by an accumulation of immunosuppressive myeloid cells and exclusion of activated T cells. Here, we demonstrate that genetic ablation or therapeutic inhibition of the myeloid-specific hematopoietic cell kinase (HCK) enables activity of antagonistic anti-programmed cell death protein 1 (anti-PD1), anti-CTLA4, or agonistic anti-CD40 immunotherapies in otherwise refractory tumors and augments response in treatment-susceptible tumors. Mechanistically, HCK ablation reprograms tumor-associated macrophages and dendritic cells toward an inflammatory endotype and enhances CD8+ T cell recruitment and activation when combined with immunotherapy in mice. Meanwhile, therapeutic inhibition of HCK in humanized mice engrafted with patient-derived xenografts counteracts tumor immunosuppression, improves T cell recruitment, and impairs tumor growth. Collectively, our results suggest that therapeutic targeting of HCK activity enhances response to immunotherapy by simultaneously stimulating immune cell activation and inhibiting the immunosuppressive tumor microenvironment.

STAT3 Signaling in Breast Cancer: Multicellular Actions and Therapeutic Potential.

Interleukin (IL)-6 family cytokines, such as IL-6 and IL-11, are defined by the shared use of the gp130 receptor for the downstream activation of STAT3 signaling and the activation of genes which contribute to the "hallmarks of cancer", including proliferation, survival, invasion and metastasis. Increased expression of these cytokines, or the ligand-specific receptors IL-6R and IL-11RA, in breast tumors positively correlate to disease progression and poorer patient outcome. In this review, we examine evidence from pre-clinical studies that correlate enhanced IL-6 and IL-11 mediated gp130/STAT3 signaling to the progression of breast cancer. Key processes by which the IL-6 family cytokines contribute to the heterogeneous nature of breast cancer, immune evasion and metastatic potential, are discussed. We examine the latest research into the therapeutic targeting of IL-6 family cytokines that inhibit STAT3 transcriptional activity as a potential breast cancer treatment, including current clinical trials. The importance of the IL-6 family of cytokines in cellular processes that promote the development and progression of breast cancer warrants further understanding of the molecular basis for its actions to help guide the development of future therapeutic targets.

Host IL11 Signaling Suppresses CD4+ T cell-Mediated Antitumor Responses to Colon Cancer in Mice.

IL11 is a member of the IL6 family of cytokines and signals through its cognate receptor subunits, IL11RA and glycoprotein 130 (GP130), to elicit biological responses via the JAK/STAT signaling pathway. IL11 contributes to cancer progression by promoting the survival and proliferation of cancer cells, but the potential immunomodulatory properties of IL11 signaling during tumor development have thus far remained unexplored. Here, we have characterized a role for IL11 in regulating CD4+ T cell-mediated antitumor responses. Absence of IL11 signaling impaired tumor growth in a sporadic mouse model of colon cancer and syngeneic allograft models of colon cancer. Adoptive bone marrow transfer experiments and in vivo depletion studies demonstrated that the tumor-promoting activity of IL11 was mediated through its suppressive effect on host CD4+ T cells in the tumor microenvironment. Indeed, when compared with Il11ra-proficient CD4+ T cells associated with MC38 tumors, their Il11ra-deficient counterparts displayed elevated expression of mRNA encoding the antitumor mediators IFNγ and TNFα. Likewise, IL11 potently suppressed the production of proinflammatory cytokines (IFNγ, TNFα, IL6, and IL12p70) by CD4+ T cells in vitro, which we corroborated by RNAscope analysis of human colorectal cancers, where IL11RAhigh tumors showed less IFNG and CD4 expression than IL11RAlow tumors. Therefore, our results ascribe a tumor cell-extrinsic immunomodulatory role to IL11 during colon cancer development that could be amenable to an anticytokine-based therapy.See related Spotlight by van der Burg, p. 724.

STAT3 Signalling via the IL-6ST/gp130 Cytokine Receptor Promotes Epithelial Integrity and Intestinal Barrier Function during DSS-Induced Colitis.

The intestinal epithelium provides a barrier against commensal and pathogenic microorganisms. Barrier dysfunction promotes chronic inflammation, which can drive the pathogenesis of inflammatory bowel disease (IBD) and colorectal cancer (CRC). Although the Signal Transducer and Activator of Transcription-3 (STAT3) is overexpressed in both intestinal epithelial cells and immune cells in IBD patients, the role of the interleukin (IL)-6 family of cytokines through the shared IL-6ST/gp130 receptor and its associated STAT3 signalling in intestinal barrier integrity is unclear. We therefore investigated the role of STAT3 in retaining epithelial barrier integrity using dextran sulfate sodium (DSS)-induced colitis in two genetically modified mouse models, to either reduce STAT1/3 activation in response to IL-6 family cytokines with a truncated gp130∆STAT allele (GP130∆STAT/+), or by inducing short hairpin-mediated knockdown of Stat3 (shStat3). Here, we show that mice with reduced STAT3 activity are highly susceptible to DSS-induced colitis. Mechanistically, the IL-6/gp130/STAT3 signalling cascade orchestrates intestinal barrier function by modulating cytokine secretion and promoting epithelial integrity to maintain a defence against bacteria. Our study also identifies a crucial role of STAT3 in controlling intestinal permeability through tight junction proteins. Thus, therapeutically targeting the IL-6/gp130/STAT3 signalling axis to promote barrier function may serve as a treatment strategy for IBD patients.

Therapeutic Targeting of the Tumour Microenvironment in Metastatic Colorectal Cancer.

Liver metastasis is the primary contributor to the death of patients with colorectal cancer. Despite the overall success of current treatments including targeted therapy, chemotherapy, and immunotherapy combinations in colorectal cancer patients, the prognosis of patients with liver metastasis remains poor. Recent studies have highlighted the importance of the tumour microenvironment and the crosstalk within that determines the fate of circulating tumour cells in distant organs. Understanding the interactions between liver resident cells and tumour cells colonising the liver opens new therapeutic windows for the successful treatment of metastatic colorectal cancer. Here we discuss critical cellular interactions within the tumour microenvironment in primary tumours and in liver metastases that highlight potential therapeutic targets. We also discuss recent therapeutic advances for the treatment of metastatic colorectal cancer.

Repurposing of drugs as STAT3 inhibitors for cancer therapy.

Drug repurposing is a valuable approach in delivering new cancer therapeutics rapidly into the clinic. Existing safety and patient tolerability data for drugs already in clinical use represent an untapped resource in terms of identifying therapeutic agents for off-label protein targets. The multicellular effects of STAT3 mediated by a range of various upstream signaling pathways make it an attractive therapeutic target with utility in a range of diseases including cancer, and has led to the development of a variety of STAT3 inhibitors. Moreover, heightened STAT3 transcriptional activation in tumor cells and within the cells of the tumor microenvironment contribute to disease progression. Consequently, there are many STAT3 inhibitors in preclinical development or under evaluation in clinical trials for their therapeutic efficacy predominantly in inflammatory diseases and cancer. Despite these advances, many challenges remain in ultimately providing STAT3 inhibitors to patients as cancer treatments, highlighting the need not only for a better understanding of the mechanisms associated with STAT3 activation, but also how various pharmaceutical agents suppress STAT3 activity in various cancers. In this review we discuss the importance of STAT3-dependent functions in cancer, review the status of compounds designed as direct-acting STAT3 inhibitors, and describe some of the strategies for repurposing of drugs as STAT3 inhibitors for cancer therapy.

Evaluating the benefits of renin-angiotensin system inhibitors as cancer treatments.

G-protein-coupled receptors (GPCRs) are the largest and most diverse group of cellular membrane receptors identified and characterized. It is estimated that 30 to 50% of marketed drugs target these receptors. The angiotensin II receptor type 1 (AT1R) is a GPCR which signals in response to systemic alterations of the peptide hormone angiotensin II (AngII) in circulation. The enzyme responsible for converting AngI to AngII is the angiotensin-converting enzyme (ACE). Specific inhibitors for the AT1R (more commonly known as AT1R blockers or antagonists) and ACE are well characterized for their effects on the cardiovascular system. Combined with the extensive clinical data available on patient tolerance of AT1R blockers (ARBs) and ACE inhibitors (ACEIs), as well as their non-classical roles in cancer, the notion of repurposing this class of medications as cancer treatment(s) is explored in the current review. Given that AngII-dependent AT1R activity directly regulates angiogenesis, remodeling of vasculature, pro-inflammatory responses, stem cell programming and hematopoiesis, and electrolyte balance; the modulation of these processes with pharmacologically well characterized medications could present a valuable complementary treatment option for cancer patients.

Inhibition of the SRC Kinase HCK Impairs STAT3-Dependent Gastric Tumor Growth in Mice.

Persistent activation of the latent transcription factor STAT3 is observed in gastric tumor epithelial and immune cells and is associated with a poor patient prognosis. Although targeting STAT3-activating upstream kinases offers therapeutically viable targets with limited specificity, direct inhibition of STAT3 remains challenging. Here we provide functional evidence that myeloid-specific hematopoietic cell kinase (HCK) activity can drive STAT3-dependent epithelial tumor growth in mice and is associated with alternative macrophage activation alongside matrix remodeling and tumor cell invasion. Accordingly, genetic reduction of HCK expression in bone marrow-derived cells or systemic pharmacologic inhibition of HCK activity suppresses alternative macrophage polarization and epithelial STAT3 activation, and impairs tumor growth. These data validate HCK as a molecular target for the treatment of human solid tumors harboring excessive STAT3 activity.

Repurposing the selective estrogen receptor modulator bazedoxifene to suppress gastrointestinal cancer growth.

Excessive signaling through gp130, the shared receptor for the interleukin (IL)6 family of cytokines, is a common hallmark in solid malignancies and promotes their progression. Here, we established the in vivo utility of bazedoxifene, a steroid analog clinically approved for the treatment of osteoporosis, to suppress gp130-dependent tumor growth of the gastrointestinal epithelium. Bazedoxifene administration reduced gastric tumor burden in gp130Y757F mice, where tumors arise exclusively through excessive gp130/STAT3 signaling in response to the IL6 family cytokine IL11. Likewise, in mouse models of sporadic colon and intestinal cancers, which arise from oncogenic mutations in the tumor suppressor gene Apc and the associated ß-catenin/canonical WNT pathway, bazedoxifene treatment reduces tumor burden. Consistent with the proposed orthogonal tumor-promoting activity of IL11-dependent gp130/STAT3 signaling, tumors of bazedoxifene-treated Apc-mutant mice retain excessive nuclear accumulation of ß-catenin and aberrant WNT pathway activation. Likewise, bazedoxifene treatment of human colon cancer cells harboring mutant APC did not reduce aberrant canonical WNT signaling, but suppressed IL11-dependent STAT3 signaling. Our findings provide compelling proof of concept to support the repurposing of bazedoxifene for the treatment of gastrointestinal cancers in which IL11 plays a tumor-promoting role.

ADAM17 is required for EGF-R-induced intestinal tumors via IL-6 trans-signaling.

Colorectal cancer is treated with antibodies blocking epidermal growth factor receptor (EGF-R), but therapeutic success is limited. EGF-R is stimulated by soluble ligands, which are derived from transmembrane precursors by ADAM17-mediated proteolytic cleavage. In mouse intestinal cancer models in the absence of ADAM17, tumorigenesis was almost completely inhibited, and the few remaining tumors were of low-grade dysplasia. RNA sequencing analysis demonstrated down-regulation of STAT3 and Wnt pathway components. Because EGF-R on myeloid cells, but not on intestinal epithelial cells, is required for intestinal cancer and because IL-6 is induced via EGF-R stimulation, we analyzed the role of IL-6 signaling. Tumor formation was equally impaired in IL-6-/- mice and sgp130Fc transgenic mice, in which only trans-signaling via soluble IL-6R is abrogated. ADAM17 is needed for EGF-R-mediated induction of IL-6 synthesis, which via IL-6 trans-signaling induces ß-catenin-dependent tumorigenesis. Our data reveal the possibility of a novel strategy for treatment of colorectal cancer that could circumvent intrinsic and acquired resistance to EGF-R blockade.

The angiotensin receptor blocker, Losartan, inhibits mammary tumor development and progression to invasive carcinoma.

Drugs that target the Renin-Angiotensin System (RAS) have recently come into focus for their potential utility as cancer treatments. The use of Angiotensin Receptor Blockers (ARBs) and Angiotensin-Converting Enzyme (ACE) Inhibitors (ACEIs) to manage hypertension in cancer patients is correlated with improved survival outcomes for renal, prostate, breast and small cell lung cancer. Previous studies demonstrate that the Angiotensin Receptor Type I (AT1R) is linked to breast cancer pathogenesis, with unbiased analysis of gene-expression studies identifying significant up-regulation of AGTR1, the gene encoding AT1R in ER+ve/HER2-ve tumors correlating with poor prognosis. However, there is no evidence, so far, of the functional contribution of AT1R to breast tumorigenesis. We explored the potential therapeutic benefit of ARB in a carcinogen-induced mouse model of breast cancer and clarified the mechanisms associated with its success.Mammary tumors were induced with 7,12-dimethylbenz[α]antracene (DMBA) and medroxyprogesterone acetate (MPA) in female wild type mice and the effects of the ARB, Losartan treatment assessed in a preventative setting (n = 15 per group). Tumor histopathology was characterised by immunohistochemistry, real-time qPCR to detect gene expression signatures, and tumor cytokine levels measured with quantitative bioplex assays. AT1R was detected with radiolabelled ligand binding assays in fresh frozen tumor samples.We showed that therapeutic inhibition of AT1R, with Losartan, resulted in a significant reduction in tumor burden; and no mammary tumor incidence in 20% of animals. We observed a significant reduction in tumor progression from DCIS to invasive cancer with Losartan treatment. This was associated with reduced tumor cell proliferation and a significant reduction in IL-6, pSTAT3 and TNFα levels. Analysis of tumor immune cell infiltrates, however, demonstrated no significant differences in the recruitment of lymphocytes or tumour-associated macrophages in Losartan or vehicle-treated mammary tumors.Analysis of AT1R expression with radiolabelled ligand binding assays in human breast cancer biopsies showed high AT1R levels in 30% of invasive ductal carcinomas analysed. Furthermore, analysis of the TCGA database identified that high AT1R expression to be associated with luminal breast cancer subtype.Our in vivo data and analysis of human invasive ductal carcinoma samples identify the AT1R is a potential therapeutic target in breast cancer, with the availability of a range of well-tolerated inhibitors currently used in clinics. We describe a novel signalling pathway critical in breast tumorigenesis, that may provide new therapeutic avenues to complement current treatments.

Insights into Orphan Nuclear Receptors as Prognostic Markers and Novel Therapeutic Targets for Breast Cancer.

There is emerging evidence asserting the importance of orphan nuclear receptors (ONRs) in cancer initiation and progression. In breast cancer, there is a lot unknown about ONRs in terms of their expression profile and their transcriptional targets in the various stages of tumor progression. With the classification of breast tumors into distinct molecular subtypes, we assess ONR expression in the different breast cancer subtypes and with patient outcomes. Complementing this, we review evidence implicating ONR-dependent molecular pathways in breast cancer progression to identify candidate ONRs as potential prognostic markers and/or as therapeutic targets.

Conditional overexpression of liver receptor homolog-1 in female mouse mammary epithelium results in altered mammary morphogenesis via the induction of TGF-ß.

Liver receptor homolog-1 (LRH-1) is an orphan nuclear receptor that belongs to the NR5A subgroup of nuclear receptors. LRH-1 induces key genes to regulate metabolic process, ovarian function, cancer cell proliferation, and steroidogenesis. In the breast, LRH-1 modulates and synergizes with endogenous estrogen signaling to promote breast cancer cell proliferation. We used small interfering RNA knockdown strategies to deplete LRH-1 in breast cancer cells and followed with microarray analysis to identify LRH-1-dependent mechanisms. We identified key genes involved in TGF-ß signaling to be highly responsive to LRH-1 knockdown. This relationship was validated in 2 breast cancer cell lines overexpressing LRH-1 in vitro and in a novel transgenic mouse with targeted LRH-1 overexpression in mammary epithelial cells. Notably, TGF-ß signaling was activated in LRH-1-overexpressing breast cancer cells and mouse mammary glands. Further analyses of mammary gross morphology revealed a significant reduction in mammary lateral budding after LRH-1 overexpression. These findings suggest that the altered mammary morphogenesis in LRH-1 transgenic animals is mediated via enhanced TGF-ß expression. The regulation of TGF-ß isoforms and SMAD2/3-mediated downstream signaling by LRH-1 also implicates a potential contribution of LRH-1 in breast cancer. Collectively, these data demonstrate that LRH-1 regulates TGF-ß expression and downstream signaling in mouse mammary glands.

Distinct nuclear receptor expression in stroma adjacent to breast tumors.

The interaction between breast tumor epithelial and stromal cells is vital for initial and recurrent tumor growth. While breast cancer-associated stromal cells provide a favorable environment for proliferation and metastasis, the molecular mechanisms contributing to this process are not fully understood. Nuclear receptors (NRs) are intracellular transcription factors that directly regulate gene expression. Little is known about the status of NRs in cancer-associated stroma. Nuclear Receptor Low-Density Taqman Arrays were used to compare the gene expression profiles of all 48 NR family members in a collection of primary cultured cancer-associated fibroblasts (CAFs) obtained from estrogen receptor (ER)α positive breast cancers (n = 9) and normal breast adipose fibroblasts (NAFs) (n = 7). Thirty-three of 48 NRs were expressed in both the groups, while 11 NRs were not detected in either. Three NRs (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1); estrogen-related receptor beta (ERR-ß); and RAR-related orphan receptor beta (ROR-ß)) were only detected in NAFs, while one NR (liver receptor homolog-1 (LRH-1)) was unique to CAFs. Of the NRs co-expressed, four were significantly down-regulated in CAFs compared with NAFs (RAR-related orphan receptor-α (ROR-α); Thyroid hormone receptor-ß (TR-ß); vitamin D receptor (VDR); and peroxisome proliferator-activated receptor-γ (PPAR-γ)). Quantitative immunohistochemistry for LRH-1, TR-ß, and PPAR-γ proteins in stromal fibroblasts from an independent panel of breast cancers (ER-positive (n = 15), ER-negative (n = 15), normal (n = 14)) positively correlated with mRNA expression profiles. The differentially expressed NRs identified in tumor stroma are key mediators in aromatase regulation and subsequent estrogen production. Our findings reveal a distinct pattern of NR expression that therefore fits with a sustained and increased local estrogen microenvironment in ER-positive tumors. NRs in CAFs may provide a new avenue for the development of intratumoral-targeted therapies in breast cancer.

Oestradiol reduces liver receptor homolog-1 mRNA transcript stability in breast cancer cell lines.

The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E2), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER- cells. However, the presence of LRH-1 protein in ER- cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER- breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER- compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E2, showed increased mRNA stability in ER- versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E2 treatment, this effect mediated by ERα. Our data demonstrates that in ER- cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER- cells as well as ER- tumors suggests a possible role in the development of ER- tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER- and ER+ breast cancer.

Liver receptor homologue-1 expression in ovarian epithelial and granulosa cell tumours.

Granulosa cell tumours of the ovary (GCT) express aromatase and produce oestrogens. The ovarian-specific aromatase promoter (pII) is regulated by members of the group 5A nuclear receptor family, SF-1 and LRH-1. Since both SF-1 and LRH-1 are implicated in proliferation and cancer, we hypothesised that alteration in the expression of either or both receptors may be associated with GCT. We therefore determined the expression of LRH-1, SF-1 and aromatase in a cohort of GCT, mucinous and serous cystadenocarcinomas, and normal ovaries. LRH-1 mRNA was present at low level in normal ovary and serous cystadenocarcinoma, but was elevated approximately 30-fold in GCT, and 8-fold in mucinous cystadenocarcinoma, compared to normal ovary. LRH-1 protein expression was confirmed in GCT by immunohistochemistry. SF-1 mRNA was significantly lower that of LRH-1 in all samples and not significantly altered in GCT, compared to normal ovary. Aromatase mRNA was present at low level in normal ovary and serous and mucinous cystadenocarcinoma, and significantly elevated (18-fold) in GCT compared to normal ovary. Despite the coordinate over-expression of both LRH-1 and aromatase in GCT versus normal ovary, their levels did not correlate in individual patients; rather, aromatase expression correlated with that of SF-1. Finally, although both LRH-1 and SF-1 activated aromatase promoter activity in transient transfection studies, gel-shift and chromatin immunoprecipitation data indicated that SF-1, but not LRH-1, bound to the aromatase promoter. We conclude that SF-1 regulates aromatase expression in GCT; over-expression of LRH-1 suggests that this receptor may be involved in the pathogenesis of GCT by mechanisms other than the regulation of aromatase. Its role in this disease therefore warrants further investigation.