GHS-R suppression in adipose tissues protects against obesity and insulin resistance by regulating adipose angiogenesis and fibrosis.

BACKGROUND/OBJECTIVES: Ghrelin is an orexigenic hormone that increases food intake, adiposity, and insulin resistance through its receptor Growth Hormone Secretagogue Receptor (GHS-R). We previously showed that ghrelin/GHS-R signaling has important roles in regulation of energy homeostasis, and global deletion of GHS-R reduces obesity and improves insulin sensitivity by increasing thermogenesis. However, it is unknown whether GHS-R regulates thermogenic activation in adipose tissues directly. METHODS: We generated a novel adipose tissue-specific GHS-R deletion mouse model and characterized the mice under regular diet (RD) and high-fat diet (HFD) feeding. Body composition was measured by Echo MRI. Metabolic profiling was determined by indirect calorimetry. Response to environmental stress was assessed using a TH-8 temperature monitoring system. Insulin sensitivity was evaluated by glucose and insulin tolerance tests. Tissue histology was analyzed by hematoxylin/eosin and immunofluorescent staining. Expression of genes involved in thermogenesis, angiogenesis and fibrosis in adipose tissues were analyzed by real-time PCR. RESULTS: Under RD feeding, adipose tissue-specific GHS-R deletion had little or no impact on metabolic parameters. However, under HFD feeding, adipose tissue-specific GHS-R deletion attenuated diet-induced obesity and insulin resistance, showing elevated physical activity and heat production. In addition, adipose tissue-specific GHS-R deletion increased expression of master adipose transcription regulator of peroxisome proliferator-activated receptor (PPAR) γ1 and adipokines of adiponectin and fibroblast growth factor (FGF) 21; and differentially modulated angiogenesis and fibrosis evident in both gene expression and histological analysis. CONCLUSIONS: These results show that GHS-R has cell-autonomous effects in adipocytes, and suppression of GHS-R in adipose tissues protects against diet-induced obesity and insulin resistance by modulating adipose angiogenesis and fibrosis. These findings suggest adipose GHS-R may constitute a novel therapeutic target for treatment of obesity and metabolic syndrome.

Intracrine VEGF signalling mediates colorectal cancer cell migration and invasion.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) are key regulators of angiogenesis, affecting endothelial cell survival and function. However, the effect of VEGF-VEGFR signalling on tumour cell function is not well understood. Our previous studies in colorectal cancer (CRC) cells have demonstrated an intracrine VEGF/VEGFR1 signalling mechanism that mediates CRC cell survival and chemo-sensitivity. Since extracellular VEGF signalling regulates migration of endothelial cells and various tumour cells, we attempted to determine whether intracrine VEGF signalling affects CRC cell motility. METHODS: Migration and invasion of CRC cells, with and without VEGF or VEGFR1 depletion, were assayed using transwell migration chambers. Changes in cell morphology, epithelial-mesenchymal transition (EMT) markers, and markers of cell motility were assessed by immunostaining and western blot. RESULTS: Depletion of intracellular VEGF and VEGFR1 in multiple CRC cell lines led to strong inhibition of migration and invasion of CRC cells. Except for Twist, there were no significant differences in markers of EMT between control and VEGF/VEGFR1-depleted CRC cells. However, VEGF/VEGFR1-depleted CRC cells demonstrated a significant reduction in levels of phosphorylated focal adhesion kinase and its upstream regulators pcMET and pEGFR. CONCLUSIONS: Inhibition of intracrine VEGF signalling strongly inhibits CRC cell migration and invasion by regulating proteins involved in cell motility.

Nutrient-Sensing Ghrelin Receptor in Macrophages Modulates Bisphenol A-Induced Intestinal Inflammation in Mice.

Bisphenols are environmental toxins with endocrine disruptor activity, yet bisphenol A (BPA) and its analogs are still widely used in manufacturing plastic products. There is evidence showing that BPA elicits inflammation in humans and animals, but the target cell types of BPA are not well understood. In this study, we sought to determine BPA's direct effect on macrophages and BPA immunotoxicity in mouse intestine. Ghrelin is an important nutrient-sensing hormone, acting through its receptor growth hormone secretagogue receptor (GHSR) to regulate metabolism and inflammation. We found that BPA promotes intestinal inflammation, showing increased infiltrating immune cells in colons and enhanced expression of Ghsr and pro-inflammatory cytokines and chemokines, such as Il6 and Ccl2, in colonic mucosa. Moreover, we found that both long- and short-term BPA exposure elevated pro-inflammatory monocytes and macrophages in mouse peripheral blood mononuclear cells (PBMC) and peritoneal macrophages (PM), respectively. To determine the role of GHSR in BPA-mediated inflammation, we generated Ghsr deletion mutation in murine macrophage RAW264.7 using CRISPR gene editing. In wild-type RAW264.7 cells, the BPA exposure promotes macrophage pro-inflammatory polarization and increases Ghsr and cytokine/chemokine Il6 and Ccl2 expression. Interestingly, Ghsr deletion mutants showed a marked reduction in pro-inflammatory cytokine/chemokine expression in response to BPA, suggesting that GHSR is required for the BPA-induced pro-inflammatory response. Further understanding how nutrient-sensing GHSR signaling regulates BPA intestinal immunotoxicity will help design new strategies to mitigate BPA immunotoxicity and provide policy guidance for BPA biosafety.

Aberrant expression of katanin p60 in prostate cancer bone metastasis.

BACKGROUND: Katanin p60 is a microtubule-severing protein and is involved in microtubule cytoskeleton organization in both mitotic and non-mitotic processes. Its role in cancer metastasis is unknown. METHODS: Differential protein profiles of bone marrow aspirates were analyzed by chromatography, electrophoresis, and mass spectrometry. Expression of katanin p60 in primary and metastatic prostate cancer was examined by immunohistochemistry. Cellular function of katanin p60 was further examined in prostate cell lines. RESULTS: In a proteomic profiling of bone marrow aspirates from men with prostate cancer, we found that katanin p60 was one of the proteins differentially expressed in bone metastasis samples. Immunohistochemical staining showed that katanin p60 was expressed in the basal cells in normal human prostate glands. In prostatic adenocarcinomas, in which the basal cells were absent, katanin p60 was expressed in the prostate cancer cells. In the specimens from bone metastasis, katanin p60 was detectable in the metastatic cancer cells. Strikingly, some of the metastatic cancer cells also co-expressed basal cell biomarkers including the tumor suppressor p53-homologous protein p63 and the high molecular weight cytokeratins, suggesting that the metastatic prostate cancer cells may have a basal cell-like phenotype. Moreover, overexpression of katanin p60 inhibited prostate cancer cell proliferation but enhanced cell migration activity. CONCLUSIONS: Katanin p60 was aberrantly expressed during prostate cancer progression. Its expression in the metastatic cells in bone was associated with the re-emergence of a basal cell-like phenotype. The elevated katanin p60 expression may contribute to cancer cell metastasis via a stimulatory effect on cell motility.

Binding Domain Characterization of Growth Hormone Secretagogue Receptor.

Background and Objectives: Activation of ghrelin receptor growth hormone secretagogue receptor (GHS-R) by endogenous or synthetic ligands amplifies pulsatile release of growth hormone (GH) and enhances food intake, very relevant to development and growth. GHS-R is a G-protein coupled receptor that has great druggable potential. Understanding the precise ligand and receptor interactions is crucial to advance the application of GHS-R. Materials and Methods: We used radiolabeled ligand-binding assay and growth hormone release assay to assess the binding and functional characteristics of GHS-R to synthetic agonists MK-0677 and GHS-25, as well as to endogenous peptide ligand ghrelin. We analyzed the ligand-dependent activity of GHS-R by measuring aequorin-based [Ca++]i responses. To define a ligand-binding pocket of GHS-R, we generated a series of human/puffer fish GHS-R chimeras by domain swapping, as well as a series of mutants by site-directed mutagenesis. Results: We found that the synthetic ligands have high binding affinity to GHS-R in the in vitro competitive binding assay. Remarkably, the in vivo GH secretagogue activity is higher with the synthetic agonists MK-0677 and GHS-25 than that of ghrelin. Importantly, the activity was completely abolished in GHS-R knockout mice. In GHS-R chimera analysis, we identified the C-terminal region, particularly the transmembrane domain 6 (TM6), to be critical for the ligand-dependent activity. Our site-directed mutagenesis study further revealed that amino acid residues D99 and W276 in GHS-R are essential for ligand binding. Interestingly, critical residues distinctively interact with different ligands, MK-0677 activation depends on E124, while ghrelin and GHS-25 preferentially interact with F279. Conclusion: The ligand-binding pocket of human GHS-R is mainly defined by interactive residues in TM6 and the adjacent region of the receptor. This novel finding in GHS-R binding domains advances the structural/ functional understanding of GHS-R, which will help to select/design better GHS-R agonists/ antagonists for future therapeutic applications.

Cytospin-A Regulates Colorectal Cancer Cell Division and Migration by Modulating Stability of Microtubules and Actin Filaments.

Proteins that interact with cytoskeletal elements play important roles in cell division and are potentially important targets for therapy in cancer. Cytospin-A (CYTSA), a protein known to interact with actin and microtubules, has been previously described to be important in various developmental disorders, including oblique facial clefting. We hypothesized that CYTSA plays an important role in colorectal cancer (CRC) cell division. The effects of CYTSA depletion on CRC cell proliferation were analyzed using cell growth assays, microscopic analyses of live and fixed cells, and time-lapse imaging. CYTSA depletion led to inhibition of cell proliferation, significant increases in CRC cell death, and accumulation of doublet cells during and following cell division. Depletion of CYTSA also resulted in strong inhibition of CRC cell migration and invasion. Mechanistically, CYTSA depletion resulted in significant decreases in the stability of microtubules and altered polymerization of actin filaments in CRC cells. Finally, bioinformatic analyses were performed to determine the correlation between CYTSA expression and survival of patients with CRC. Interestingly, a strong correlation between high CYTSA expression and poor survival was observed in the TCGA adenocarcinoma data set but not in an independent data set. Since inhibiting CYTSA significantly reduces CRC cell proliferation, migration, and invasion, targeting CYTSA may be a potential novel therapeutic option for patients with metastatic CRC.

Androgen depletion up-regulates cadherin-11 expression in prostate cancer.

Men with castration-resistant prostate cancer (PCa) frequently develop metastasis in bone. The reason for this association is unclear. We have previously shown that cadherin-11 (also known as OB-cadherin), a homophilic cell adhesion molecule that mediates osteoblast adhesion, plays a role in the metastasis of PCa to bone. Here, we report that androgen-deprivation therapy up-regulates cadherin-11 expression in PCa. In human PCa specimens, immunohistochemical staining showed that 22/26 (85%) primary PCa tumours from men with castration-resistant PCa expressed cadherin-11. In contrast, only 7/50 (14%) androgen-dependent PCa tumours expressed cadherin-11. In the MDA-PCa-2b xenograft animal model, cadherin-11 was expressed in the recurrent tumours following castration. In the PCa cell lines, there is an inverse correlation between expression of cadherin-11 and androgen receptor (AR), and cadherin-11 is expressed in very low levels or not expressed in AR-positive cell lines, including LNCaP, C4-2B4 and VCaP cells. We showed that AR likely regulates cadherin-11 expression in PCa through an indirect mechanism. Although re-expression of AR in the AR-negative PC3 cells led to the inhibition of cadherin-11 expression, depletion of androgen from the culture medium or down-regulation of AR by RNA interference in the C4-2B4 cells or VCaP cells only produced a modest increase of cadherin-11 expression. Promoter analysis indicated that cadherin-11 promoter does not contain a typical AR-binding element, and AR elicits a modest inhibition of cadherin-11 promoter activity, suggesting that AR does not regulate cadherin-11 expression directly. Together, these results suggest that androgen deprivation up-regulates cadherin-11 expression in prostate cancer, and this may contribute to the metastasis of PCa to bone. Our study suggests that therapeutic strategies that block cadherin-11 expression or function may be considered when applying androgen-ablation therapy.

GHS-R in brown fat potentiates differential thermogenic responses under metabolic and thermal stresses.

In response to cold or diet, fatty acids are dissipated into heat through uncoupling protein 1 (UCP1) in brown adipose tissue (BAT). This process is termed non-shivering thermogenesis, which is important for body temperature maintenance and contributes to obesity pathogenesis. Thermogenic enhancement has been considered a promising anti-obesity strategy. Ghrelin and its receptor Growth Hormone Secretagogue Receptor (GHS-R) have critical roles in energy intake, nutrient sensing, and lipid metabolism. We previously reported that global Ghsr-knockout mice have increased energy expenditure due to enhanced thermogenesis. To determine the site of action for GHS-R mediated thermogenesis, we generated brown adipocyte-specific Ghsr knockout mice (UCP1-CreER/Ghsrf/f) and assessed thermogenic responses under regular diet (RD) fed homeostatic metabolic state or high-fat diet (HFD) fed metabolically-impaired obese state, under normal or cold housing environment. Under a RD-feeding, UCP1-CreER/Ghsrf/f mice showed increased body fat and a slightly elevated core body temperature under cold but not under normal temperature. Consistently, the expression of thermogenic genes in BAT of RD-fed UCP1-CreER/Ghsrf/f mice was increased in reposes to cold. Under HFD feeding, HFD-fed UCP1-CreER/Ghsrf/f mice showed no difference in body fat or body temperature under either normal or cold exposure. Interestingly, the expression of thermogenic genes in BAT of HFD-fed UCP1-CreER/Ghsrf/f mice was upregulated under normal temperature but downregulated under cold exposure. Overall, our data show that GHS-R has cell-autonomous effect in brown adipocytes, and GHS-R regulates BAT thermogenic activity in a temperature- and metabolic state-dependent manner. The thermogenic effect of GHS-R in BAT is more pronounced in cold environment and differentially variable based on metabolic state; under cold exposure, GHS-R inhibition in BAT activates thermogenesis under homeostatic state but suppresses thermogenesis under obese state. Our finding collectively suggests that GHS-R in BAT, acting as a "metabolic thermostat", differentially regulates thermogenesis in response to different metabolic and thermal stimuli.

Cadherin-11 promotes the metastasis of prostate cancer cells to bone.

Bone is the most common site of metastases from prostate cancer. The mechanism by which prostate cancer cells metastasize to bone is not fully understood, but interactions between prostate cancer cells and bone cells are thought to initiate the colonization of metastatic cells at that site. Here, we show that cadherin-11 (also known as osteoblast-cadherin) was highly expressed in prostate cancer cell line derived from bone metastases and had strong homophilic binding to recombinant cadherin-11 in vitro. Down-regulation of cadherin-11 in bone metastasis-derived PC3 cells with cadherin-11-specific short hairpin RNA (PC3-shCad-11) significantly decreased the adhesion of those cells to cadherin-11 in vitro. In a mouse model of metastasis, intracardiac injection of PC3 cells led to metastasis of those cells to bone. However, the incidence of PC3 metastasis to bone in this model was reduced greatly when the expression of cadherin-11 by those cells was silenced. The clinical relevance of cadherin-11 in prostate cancer metastases was further studied by examining the expression of cadherin-11 in human prostate cancer specimens. Cadherin-11 was not expressed by normal prostate epithelial cells but was detected in prostate cancer, with its expression increasing from primary to metastatic disease in lymph nodes and especially bone. Cadherin-11 expression was not detected in metastatic lesions that occur in other organs. Collectively, these findings suggest that cadherin-11 is involved in the metastasis of prostate cancer cells to bone.

Soluble ErbB3 levels in bone marrow and plasma of men with prostate cancer.

PURPOSE: Prostate cancer tends to metastasize to bone and induce osteoblastic lesions. We identified a soluble form of ErbB3 (sErbB3), p45-sErbB3, in bone marrow supernatant from men with prostate cancer bone metastasis and showed that p45-sErbB3 enhances bone formation. We aimed to understand clinical implications of sErbB3 by establishing an ELISA to detect sErbB3 levels in bone marrow and plasma samples. EXPERIMENTAL DESIGN: We did ELISAs on marrow from 108 men [34 with androgen-dependent disease, 30 with androgen-independent disease (AI) but negative bone scan (AI/BS-), and 44 with AI and positive bone scan (AI/BS+)], sequential marrow from 5 men during treatment, plasma from 52 men before and after docetaxel treatment, and plasma from 95 men ages > or =70 years old without prostate cancer. RESULTS: Some men with clinically detectable bone metastasis had high sErbB3 levels. Within the AI/BS- group, higher sErbB3 levels seemed to yield lower rates of bone metastasis. In the AI/BS+ group, detectable bone metastases took longer to appear in men with higher sErbB3 levels than in men with lower sErbB3 levels (median, 82 versus 41 months). However, high sErbB3 levels did not confer survival benefit after metastasis development. Among men with metastatic progression in bone, docetaxel treatment reduced plasma sErbB3 (P < 0.0001) but did not affect bone-specific alkaline phosphatase (P = 0.206) or prostate-specific antigen (P = 0.906). sErbB3 was also detected in men without prostate cancer. CONCLUSIONS: The apparent correlation between higher sErbB3 levels and longer time to bone metastasis suggests that sErbB3 participates in progression in bone of prostate cancer.

A secreted isoform of ErbB3 promotes osteonectin expression in bone and enhances the invasiveness of prostate cancer cells.

The propensity for prostate cancer to metastasize to bone led us and others to propose that bidirectional interactions between prostate cancer cells and bone are critical for the preferential metastasis of prostate cancer to bone. We identified previously a secreted isoform of ErbB3 (p45-sErbB3) in bone marrow supernatant samples from men with prostate cancer and bone metastasis and showed by immunohistochemical analysis of human tissue specimens that p45-sErbB3 was highly expressed in metastatic prostate cancer cells in bone. Here, we show that p45-sErbB3 stimulated mouse calvaria to secrete factors that increased the invasiveness of prostate cancer cells in a Boyden chamber invasion assay. Using gene array analysis to identify p45-sErbB3-responsive genes, we found that p45-sErbB3 up-regulated the expression of osteonectin/SPARC, biglycan, and type I collagen in calvaria. We further show that recombinant osteonectin increased the invasiveness of PC-3 cells, whereas osteonectin-neutralizing antibodies blocked this p45-sErbB3-induced invasiveness. These results indicate that p45-sErbB3 enhances the invasiveness of PC-3 cells in part by stimulating the secretion of osteonectin by bone. Thus, p45-sErbB3 may mediate the bidirectional interactions between prostate cancer cells and bone via osteonectin.

Endothelial Cells Promote Colorectal Cancer Cell Survival by Activating the HER3-AKT Pathway in a Paracrine Fashion.

The regulation of colorectal cancer cell survival pathways remains to be elucidated. Previously, it was demonstrated that endothelial cells (EC) from the liver (liver parenchymal ECs or LPEC), the most common site of colorectal cancer metastases, secrete soluble factors in the conditioned medium (CM) that, in turn, increase the cancer stem cell phenotype in colorectal cancer cells. However, the paracrine effects of LPECs on other colorectal cancer cellular functions have not been investigated. Here, results showed that CM from LPECs increased cell growth and chemoresistance by activating AKT in colorectal cancer cells in vitro. Using an unbiased receptor tyrosine kinase array, it was determined that human epidermal growth factor receptor 3 (ERBB3/HER3) was activated by CM from LPECs, and it mediated AKT activation, cell growth, and chemoresistance in colorectal cancer cells. Inhibition of HER3, either by an inhibitor AZD8931 or an antibody MM-121, blocked LPEC-induced HER3-AKT activation and cell survival in colorectal cancer cells. In addition, CM from LPECs increased in vivo tumor growth in a xenograft mouse model. Furthermore, inhibiting HER3 with AZD8931 significantly blocked tumor growth induced by EC CM. These results demonstrated a paracrine role of liver ECs in promoting cell growth and chemoresistance via activating HER3-AKT in colorectal cancer cells. IMPLICATIONS: This study suggested a potential of treating patients with metastatic colorectal cancer with HER3 antibodies/inhibitors that are currently being assessed in clinical trials for various cancer types.

Bone microenvironment and androgen status modulate subcellular localization of ErbB3 in prostate cancer cells.

ErbB-3, an ErbB receptor tyrosine kinase, has been implicated in the pathogenesis of several malignancies, including prostate cancer. We found that ErbB-3 expression was up-regulated in prostate cancer cells within lymph node and bone metastases. Despite being a plasma membrane protein, ErbB-3 was also detected in the nuclei of the prostate cancer cells in the metastatic specimens. Because most metastatic specimens were from men who had undergone androgen ablation, we examined the primary tumors from patients who have undergone hormone deprivation therapy and found that a significant fraction of these specimens showed nuclear localization of ErbB3. We thus assessed the effect of androgens and the bone microenvironment on the nuclear translocation of ErbB-3 by using xenograft tumor models generated from bone-derived prostate cancer cell lines, MDA PCa 2b, and PC-3. In subcutaneous tumors, ErbB-3 was predominantly in the membrane/cytoplasm; however, it was present in the nuclei of the tumor cells in the femur. Castration of mice bearing subcutaneous MDA PCa 2b tumors induced a transient nuclear translocation of ErbB-3, with relocalization to the membrane/cytoplasm upon tumor recurrence. These findings suggest that the bone microenvironment and androgen status influence the subcellular localization of ErbB-3 in prostate cancer cells. We speculate that nuclear localization of ErbB-3 may aid prostate cancer cell survival during androgen ablation and progression of prostate cancer in bone.

A Survey on Data Reproducibility and the Effect of Publication Process on the Ethical Reporting of Laboratory Research.

Purpose: The successful translation of laboratory research into effective therapies is dependent upon the validity of peer-reviewed publications. However, several publications in recent years suggested that published scientific findings could be reproduced only 11% to 45% of the time. Multiple surveys attempted to elucidate the fundamental causes of data irreproducibility and underscored potential solutions, more robust experimental designs, better statistics, and better mentorship. However, no prior survey has addressed the role of the review and publication process on honest reporting.Experimental Design: We developed an anonymous online survey intended for trainees involved in bench research. The survey included questions related to mentoring/career development, research practice, integrity, and transparency, and how the pressure to publish and the publication process itself influence their reporting practices.Results: Responses to questions related to mentoring and training practices were largely positive, although an average of approximately 25% did not seem to receive optimal mentoring. A total of 39.2% revealed having been pressured by a principle investigator or collaborator to produce "positive" data. About 62.8% admitted that the pressure to publish influences the way they report data. The majority of respondents did not believe that extensive revisions significantly improved the manuscript while adding to the cost and time invested.Conclusions: This survey indicates that trainees believe that the pressure to publish affects honest reporting, mostly emanating from our system of rewards and advancement. The publication process itself affects faculty and trainees and appears to influence a shift in their ethics from honest reporting ("negative data") to selective reporting, data falsification, or even fabrication. Clin Cancer Res; 24(14); 3447-55. ©2018 AACR.

Macrophage conditioned medium promotes colorectal cancer stem cell phenotype via the hedgehog signaling pathway.

BACKGROUND: There is conflicting data on the role of macrophages in colorectal cancer (CRC); some studies have shown that macrophages can exert an anti-tumor effect whereas others show that macrophages are tumor promoting. We sought to determine the role of conditioned medium (CM) from macrophages, in particular classically activated macrophages, on the development of the CSC phenotype in CRC cells, which is believed to mediate tumor growth and chemoresistance. METHODS: Murine (CT26) and human (HCP-1) CRC cell lines were treated with CM from lipopolysaccharide (LPS)-activated murine macrophages. The CSC population was assessed using the sphere-forming assay and aldehyde dehydrogenase assay. Chemoresistance studies were performed using the MTT assay. CSC transcription factors and SHH protein were analyzed by Western blotting. RESULTS: The results showed that LPS-activated macrophage CM induced the CSC phenotype in CRC cells. Further studies showed that the CSC phenotype was mediated by the sonic hedgehog (SHH)-Gli signaling pathway, which is known to drive self-renewal; these effects were blocked by depletion of SHH in macrophage CM. In addition, LPS-activated macrophage CM enhanced chemoresistance. CONCLUSIONS: LPS-activated macrophages play an active role in promoting the CSC phenotype through activation of the SHH-Gli signaling pathway in CRC cells.

Biology and clinical management of prostate cancer bone metastasis.

Prostate cancer is the most common cancer among men in the United States. Advanced prostate cancer has a particular propensity to metastasize to bone, where it produces predominantly osteoblastic lesions and local bone formation. The tropism for bone is thought to be due in part to specific interactions between the prostate cancer cells and cells present in the bone environment, particularly the bone marrow endothelial cells and osteoblasts. Such interactions involve numerous signaling pathways that could serve as targets for new therapeutic agents. Because androgen directly influences the proliferation and metastasis of prostate cancer cells, the current first-line treatment for metastatic prostate cancer is androgen deprivation therapy. Subsequent therapies include chemotherapy and radiation therapy. New molecular therapies are being developed to target specific steps in the metastatic process. However, as yet none of these therapies has radically improved survival. Nonetheless, it is hoped that with better understanding of the biology of the disease, combination therapy that addresses multiple pathways that support the progression of prostate cancer in bone could significantly improve the survival and quality of life of men with prostate cancer.

Endothelial cells activate the cancer stem cell-associated NANOGP8 pathway in colorectal cancer cells in a paracrine fashion.

In colorectal cancer (CRC), cancer stem cells (CSCs) have been hypothesized to mediate cell survival and chemoresistance. Previous studies from our laboratory described a role for liver parenchymal endothelial cells (LPECs) in mediating the CSC phenotype in CRC cells in a paracrine/angiocrine fashion. The objectives of this study were to determine whether endothelial cells (ECs) from different organs can induce the CSC phenotype in CRC cells and to elucidate the signaling pathways involved. We treated a newly developed CRC cell line (HCP-1) and established CRC cell lines (HT29 and SW480) with conditioned medium (CM) from primary ECs isolated from nonmalignant liver, lung, colon mucosa, and kidney. Our results showed that CM from ECs from all organs increased the number of CSCs, as determined by sphere formation, and protein levels of NANOG and OCT4 in CRC cells. With the focus of further elucidating the role of the liver vascular network in mediating the CSC phenotype, we demonstrated that CM from LPECs increased resistance to 5-fluorouracil in CRC cells. Moreover, we showed that LPEC CM specifically induced NANOGP8 expression in CRC cells by specific enzyme digestion and a luciferase reporter assay using a vector containing the NANOGP8 promoter. Lastly, we found that LPEC CM-induced NANOGP8 expression and sphere formation were mediated by AKT activation. Our studies demonstrated a paracrine role for ECs in regulating the CSC phenotype and chemoresistance in CRC cells by AKT-mediated induction of NANOGP8. These studies suggest a more specific approach to target CSCs by blocking the expression of NANOGP8 in cancer cells.

Fusobacterium Nucleatum Subspecies Animalis Influences Proinflammatory Cytokine Expression and Monocyte Activation in Human Colorectal Tumors.

Chronic infection and associated inflammation have long been suspected to promote human carcinogenesis. Recently, certain gut bacteria, including some in the Fusobacterium genus, have been implicated in playing a role in human colorectal cancer development. However, the Fusobacterium species and subspecies involved and their oncogenic mechanisms remain to be determined. We sought to identify the specific Fusobacterium spp. and ssp. in clinical colorectal cancer specimens by targeted sequencing of Fusobacterium 16S ribosomal RNA gene. Five Fusobacterium spp. were identified in clinical colorectal cancer specimens. Additional analyses confirmed that Fusobacterium nucleatum ssp. animalis was the most prevalent F. nucleatum subspecies in human colorectal cancers. We also assessed inflammatory cytokines in colorectal cancer specimens using immunoassays and found that expression of the cytokines IL17A and TNFα was markedly increased but IL21 decreased in the colorectal tumors. Furthermore, the chemokine (C-C motif) ligand 20 was differentially expressed in colorectal tumors at all stages. In in vitro co-culture assays, F. nucleatum ssp. animalis induced CCL20 protein expression in colorectal cancer cells and monocytes. It also stimulated the monocyte/macrophage activation and migration. Our observations suggested that infection with F. nucleatum ssp. animalis in colorectal tissue could induce inflammatory response and promote colorectal cancer development. Further studies are warranted to determine if F. nucleatum ssp. animalis could be a novel target for colorectal cancer prevention and treatment. Cancer Prev Res; 10(7); 398-409. ©2017 AACR.

A Disintegrin and Metalloproteinase Domain 17 Regulates Colorectal Cancer Stem Cells and Chemosensitivity Via Notch1 Signaling.

Evidence is accumulating for the role of cancer stem cells (CSCs) in mediating chemoresistance in patients with metastatic colorectal cancer (mCRC). A disintegrin and metalloproteinase domain 17 (ADAM17; also known as tumor necrosis factor-α-converting enzyme [TACE]) was shown to be overexpressed and to mediate cell proliferation and chemoresistance in CRC cells. However, its role in mediating the CSC phenotype in CRC has not been well-characterized. The objective of the present study was to determine whether ADAM17 regulates the CSC phenotype in CRC and to elucidate the downstream signaling mechanism that mediates cancer stemness. We treated established CRC cell lines and a newly established human CRC cell line HCP-1 with ADAM17-specific small interfering RNA (siRNA) or the synthetic peptide inhibitor TAPI-2. The effects of ADAM17 inhibition on the CSC phenotype and chemosensitivity to 5-fluorouracil (5-FU) in CRC cells were examined. siRNA knockdown and TAPI-2 decreased the protein levels of cleaved Notch1 (Notch1 intracellular domain) and HES-1 in CRC cells. A decrease in the CSC phenotype was determined by sphere formation and ALDEFLUOR assays. Moreover, TAPI-2 sensitized CRC cells to 5-FU by decreasing cell viability and the median lethal dose of 5-FU and increasing apoptosis. We also showed the cleavage and release of soluble Jagged-1 and -2 by ADAM17 in CRC cells. Our studies have elucidated a role of ADAM17 in regulating the CSC phenotype and chemoresistance in CRC cells. The use of drugs that inhibit ADAM17 activity might increase the therapeutic benefit to patients with mCRC and, potentially, those with other solid malignancies.

Intracrine VEGF Signaling Mediates the Activity of Prosurvival Pathways in Human Colorectal Cancer Cells.

The effects of vascular endothelial growth factor-A (VEGF-A/VEGF) and its receptors on endothelial cells function have been studied extensively, but their effects on tumor cells are less well defined. Studies of human colorectal cancer cells where the VEGF gene has been deleted suggest an intracellular role of VEGF as a cell survival factor. In this study, we investigated the role of intracrine VEGF signaling in colorectal cancer cell survival. In human colorectal cancer cells, RNAi-mediated depletion of VEGF decreased cell survival and enhanced sensitivity to chemotherapy. Unbiased reverse phase protein array studies and subsequent validation experiments indicated that impaired cell survival was a consequence of disrupted AKT and ERK1/2 (MAPK3/1) signaling, as evidenced by reduced phosphorylation. Inhibition of paracrine or autocrine VEGF signaling had no effect on phospho-AKT or phospho-ERK1/2 levels, indicating that VEGF mediates cell survival via an intracellular mechanism. Notably, RNAi-mediated depletion of VEGF receptor VEGFR1/FLT1 replicated the effects of VEGF depletion on phospho-AKT and phospho-ERK1/2 levels. Together, these studies show how VEGF functions as an intracrine survival factor in colorectal cancer cells, demonstrating its distinct role in colorectal cancer cell survival. Cancer Res; 76(10); 3014-24. ©2016 AACR.