p-STAT3 Pathway Research Articles

Abstract 1209: FGFR inhibition re-sensitizes BRAF/MEK dual resistant cells to the BRAF/MEK inhibitor combination

Abstract The BRAF-MEK pathway is frequently mutated in human melanoma. Inhibitors of these kinases have proven to prolong survival in melanoma patients. However, clinical benefit is relatively short-lived due to acquired drug resistance. To explore mechanisms of resistance in melanoma cells treated with dual BRAF and MEK inhibitors, A375, a cell line harboring the BRAF V600E mutation, was treated with escalating doses of vemurafenib and cobimetinib to generate a cellular population resistant to combination therapy. These cells display a reversible resistance phenotype. Upon initial treatment of the drug combination, upregulation of pSTAT3 is observed but the cells lose dependency on the pSTAT3 pathway upon prolonged drug treatment. A pharmacological synthetic lethal screen was performed and the FGFR inhibitor class re-sensitizes the cells to BRAF and MEK inhibition through inhibitions of pERK. The dual resistant cells also displayed hyperactivation of the PI3K-AKT pathway. Interrogation of patient samples confirmed these findings. These findings provides a biological rationale for a polytherapy strategy using a FGFR inhibitor in patients with resistance to dual BRAF and MEK inhibition. <!–EndFragment–> Citation Format: Victoria E. Wang, Jeffrey Settleman, Frank McCormic. FGFR inhibition re-sensitizes BRAF/MEK dual resistant cells to the BRAF/MEK inhibitor combination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1209. doi:10.1158/1538-7445.AM2017-1209

MiR-138 promotes migration and tube formation of human cytomegalovirus-infected endothelial cells through the SIRT1/p-STAT3 pathway.

Human cytomegalovirus (HCMV) has been reported to be linked to vascular disease through the induction of neovessel formation. We have previously reported that microRNA (miR)-217 and miR-199a-5p enhance endothelial angiogenesis via inhibition of sirtuin 1 (SIRT1) in HCMV-infected human umbilical vein endothelial cells (HUVECs). Here, we found that miR-138 also suppressed the expression of the SIRT1 protein and stimulated phosphorylation of signal transducer and activator of transcription 3 (p-STAT3). Moreover, the regulation of p-STAT3 expression mediated by SIRT1 was found to promote HCMV-induced angiogenesis. These findings revealed that miR-138 might promote angiogenesis of HCMV-infected HUVECs by activating the SIRT1-mediated p-STAT3 pathway, and this could provide novel insights into HCMV-induced angiogenesis.

Evolution of Cancer Stem-like Cells in Endocrine-Resistant Metastatic Breast Cancers Is Mediated by Stromal Microvesicles.

The hypothesis that microvesicle-mediated miRNA transfer converts noncancer stem cells into cancer stem cells (CSC) leading to therapy resistance remains poorly investigated. Here we provide direct evidence supporting this hypothesis, by demonstrating how microvesicles derived from cancer-associated fibroblasts (CAF) transfer miR-221 to promote hormonal therapy resistance (HTR) in models of luminal breast cancer. We determined that CAF-derived microvesicles horizontally transferred miR-221 to tumor cells and, in combination with hormone therapy, activated an ERlo/Notchhi feed-forward loop responsible for the generation of CD133hi CSCs. Importantly, microvesicles from patients with HTR metastatic disease expressed high levels of miR-221. We further determined that the IL6-pStat3 pathway promoted the biogenesis of onco-miR-221hi CAF microvesicles and established stromal CSC niches in experimental and patient-derived breast cancer models. Coinjection of patient-derived CAFs from bone metastases led to de novo HTR tumors, which was reversed with IL6R blockade. Finally, we generated patient-derived xenograft (PDX) models from patient-derived HTR bone metastases and analyzed tumor cells, stroma, and microvesicles. Murine and human CAFs were enriched in HTR tumors expressing high levels of CD133hi cells. Depletion of murine CAFs from PDX restored sensitivity to HT, with a concurrent reduction of CD133hi CSCs. Conversely, in models of CD133neg, HT-sensitive cancer cells, both murine and human CAFs promoted de novo HT resistance via the generation of CD133hi CSCs that expressed low levels of estrogen receptor alpha. Overall, our results illuminate how microvesicle-mediated horizontal transfer of genetic material from host stromal cells to cancer cells triggers the evolution of therapy-resistant metastases, with potentially broad implications for their control. Cancer Res; 77(8); 1927-41. ©2017 AACR.

Changes on hepatic lipidome and HDL-lipoproteins phenotype of Wistar rats exposed chronically to cadmium

The abnormal elevation of serum PRL, referred to as hyperprolactinemia (HyperPRL), produces alterations in several reproductive parameters of male rats such as penile erection or decreased tendency to reach ejaculation. Additionally, this situation produces a significant modification of prostate histology, as observed in the epithelial structure and alveolar area, which could reach a level of hyperplasia in the long-term. In this tissue, HyperPRL produces an increase in expression of PRL receptors and activation of the Stat3 signaling pathway that is correlated with the evolution of prostate pathologies. However, the impact of HyperPRL in long-term sexually active male rats is unknown. In this work, using constantly copulating Wistar male rats with induced HyperPRL, we analyzed the level of serum PRL, the effect on prostate PRL receptors, and activation of pStat3, pStat5 and Mapk signaling pathways. Two procedures to induce HyperPRL were employed, comprising daily IP administration or adenohypophysis transplant, and although neither affected the execution of sexual behavior, the serum PRL profile following successive ejaculations was affected. Messenger RNA expression of the short and long isoforms of the PRL receptor at the ventral prostate was affected in different ways depending on the procedure to induce HyperPRL. The ventral prostate did not show any modification in terms of activation of the pStat5 signaling pathway in subjects with daily administration of PRL, although this was significantly increased in ADH transplanted subjects in the second and fourth consecutive ejaculation. A similar profile was found for the pStat3 pathway which additionally showed a significant increase in the third and fourth ejaculation of daily-injected subjects. The Mapk signaling pathway did not show any modifications in subjects with daily administration of PRL, but showed a significant increase in the second and third ejaculations of subjects with ADH transplants. Thus, although sexual behavior was not modified, HyperPRL modified the expression of PRL receptors and the activation of signal pathways in the prostate tissue. Hence, it is probable that prostatic alterations precede the sexual behavioral deficits observed in subjects with HyperPRL.

Effect of hyperprolactinemia on PRL-receptor expression and activation of Stat and Mapk cell signaling in the prostate of long-term sexually-active rats

The abnormal elevation of serum PRL, referred to as hyperprolactinemia (HyperPRL), produces alterations in several reproductive parameters of male rats such as penile erection or decreased tendency to reach ejaculation. Additionally, this situation produces a significant modification of prostate histology, as observed in the epithelial structure and alveolar area, which could reach a level of hyperplasia in the long-term. In this tissue, HyperPRL produces an increase in expression of PRL receptors and activation of the Stat3 signaling pathway that is correlated with the evolution of prostate pathologies. However, the impact of HyperPRL in long-term sexually active male rats is unknown. In this work, using constantly copulating Wistar male rats with induced HyperPRL, we analyzed the level of serum PRL, the effect on prostate PRL receptors, and activation of pStat3, pStat5 and Mapk signaling pathways. Two procedures to induce HyperPRL were employed, comprising daily IP administration or adenohypophysis transplant, and although neither affected the execution of sexual behavior, the serum PRL profile following successive ejaculations was affected. Messenger RNA expression of the short and long isoforms of the PRL receptor at the ventral prostate was affected in different ways depending on the procedure to induce HyperPRL. The ventral prostate did not show any modification in terms of activation of the pStat5 signaling pathway in subjects with daily administration of PRL, although this was significantly increased in ADH transplanted subjects in the second and fourth consecutive ejaculation. A similar profile was found for the pStat3 pathway which additionally showed a significant increase in the third and fourth ejaculation of daily-injected subjects. The Mapk signaling pathway did not show any modifications in subjects with daily administration of PRL, but showed a significant increase in the second and third ejaculations of subjects with ADH transplants. Thus, although sexual behavior was not modified, HyperPRL modified the expression of PRL receptors and the activation of signal pathways in the prostate tissue. Hence, it is probable that prostatic alterations precede the sexual behavioral deficits observed in subjects with HyperPRL.

Anandamide drives cell cycle progression through CB1 receptors in a rat model of synchronized liver regeneration.

The endocannabinoid system, through cannabinoid receptor signaling by endocannabinoids, is involved in a wide range of functions and physiopathological conditions. To date, very little is known concerning the role of the endocannabinoids in the control and regulation of cell proliferation. An anti-proliferative action of CB1 signaling blockade in neurogenesis and angiogenesis argues in favor of proliferation-promoting functions of endocannabinoids through CB1 receptors when pro-growth signals are present. Furthermore, liver regeneration, a useful in vivo model of synchronized cell proliferation, is characterized by a peak of anandamide that elicits through CB1 receptor, the expression of critical mitosis genes. The aim of this study was to focus on the timing of endocannabinoid signaling changes during the different phases of the cell cycle, exploiting the rat liver regeneration model following partial hepatectomy, the most useful to study synchronized cell cycle in vivo. Hepatic regeneration led to increased levels of anandamide and endocannabinoid-like molecules oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in the G1 phase of the cell cycle, with a concomitant increase in CB1 mRNA levels, whose protein expression peaked later during the S phase. Blocking of CB1 receptor with a low dose of the selective antagonist/inverse agonist SR141716 (0.7 mg/kg/dose) affected cell cycle progression reducing the expression of PCNA, and through the inhibition of pERK and pSTAT3 pathways. These results support the notion that the signaling mediated by anandamide through CB1 receptor may be important for the entry and progression of cells into the cell cycle and hence for their proliferation under mitogenic signals.

Impact of Inflammation–Metaplasia–Adenocarcinoma Sequence and Inflammatory Microenvironment in Esophageal Carcinogenesis Using Surgical Rat Models

Chronic inflammation has been demonstrated to correlate with tumor onset and progression. Tumor-associated macrophages (TAMs) play an important role in inflammatory tumor microenvironment. We hypothesized that an inflammatory microenvironment around TAMs may promote the development of esophageal carcinomas when induced by duodenal content reflux without carcinogens. A total gastrectomy followed by esophagojejunostomy was performed on rats in order to induce chronic duodenal content reflux esophagitis. The animals were sacrificed sequentially, at the 20th, 30th, 40th and 50th week after surgery, and their esophagi were examined. The primary antibodies against CD68, CD163, pStat3 and Foxp3 were used. Expression and localization of infiltrated cells was assessed by immunohistochemical analysis. At 20-weeks' post-surgery, squamous proliferative hyperplasia (PHP) and Barrett's metaplasia (BM) were observed. Adenocarcinoma (ADC) associated with BM, and squamous cell carcinoma (SCC) were observed 30-50weeks' post-surgery. Numerous CD68 and pStat3-positive cells were identified surrounding PHP and BM after 20weeks, and around ADC and SCC after 30weeks. Moderate infiltration of CD163-positive macrophages was seen with BM, ADC, and SCC after 30weeks. However, very few Foxp3-positive cells were observed around ADC and SCC. Macrophages infiltrate the esophagus and activate the pStat3 pathway in stromal cells and epithelium. M2 phenotype macrophages infiltrate following infiltration of M1 macrophage and contribute to tumor development through regulatory T cells (Tregs). The involvement of immune cells such as TAMs and Tregs in the inflammatory microenvironment promotes esophageal carcinogenesis.

Abstract B48: Do the gut flora play a role in the high incidence of colon cancer in African Americans?

Abstract Colonic adenomatous polyps are common tumors occurring in 50% of Western populations with 10% risk of malignant progression. To date, the environmental exposures proposed to promote the development of colorectal cancer (CRC) have been primarily dietary agents. However, the local environment to which the colonic mucosa is exposed is created by the bacterial microbiota of the colon and their metabolic products that include toxins. African Americans are known to have a higher incidence of colon cancer than other groups. We analyzed the gut flora from African Americans patients by both 16S rDNA sequencing and HIT (Human Intestinal Tract) Chip. Our findings reflected a high prevalence of Bacteroidetes and Clostridia groups with the Bacteroidetes slightly more represented in patients with colon polyps than healthy ones. While Bifidobacteria were not detected at all in all samples, Lactobacilliwere present at a very low frequency. Both bacteria are referred to as “good bacteria” and are generally associated with healthy colon. Helicobacter bacteria, associated with gastric cancer, were also detected in all analysed samples. Bacteroides fragilis and relatives were present in all samples at a high frequency. It is noteworthy that an enterotoxigenic Bacteroides fragilis have been found to trigger colon polyps formation in Min mice through a pSTAT3 pathway. A tissue microarray immunohistochemistry experiment on colon adenoma and matched normals revealed a strong expression of pSTAT3 both in colonic epithelial cells and in colonic immune cells in adenoma samples but not as much in normal ones. These findings highlight the presence of a possible ETBF-like induction mechanism in the analysed samples. Enzyme immunoassay for the detection of antibodies against ETBF toxin would confirm such findings and a metagenomic analysis of the analysed samples would shed light on predominant bacterial functions in adenoma patients that might play a role in colon oncogenic transformation in this population. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B48.

Abstract 5322: Glioma cancer stem cells induce immune suppressive macrophages

Abstract Introduction: The tumor microenvironment can induce monocytes to become immune suppressive macrophages (M2) via the phosphorylation of the signal transducer activator of transcription 3 (STAT3). The M2 cells assume a phenotype with impaired anti-tumor activity, support tumor progression, angiogenesis, and invasion and portend a poor prognosis. Gliomas contain cancer stem cells (gCSCs) that are self-renewing, multipotent, recapitulate the phenotype of the original tumor, and mediate resistance to radiation and chemotherapy. The gCSCs inhibit adaptive immune responses by inhibiting T cell proliferation and effector responses, inducing T cell apoptosis and inducing Tregs secondary to phosphorylated STAT3 (p-STAT3) regulated processes such as secreted TGF-β1 and Galectin-3 and the expression of cell surface molecules such as B7-H1. We therefore hypothesized that gCSCs also exert immune suppression of the innate immune system by inducing M2 cells. Methods: The gCSCs (n=4) were isolated from surgical glioblastoma specimens. Normal microglia were isolated from brain specimens obtained during resections of extra-axial lesions. Peripheral blood monocytes were isolated from healthy donors and differentiated into macrophages (MDMs), which were then treated with gCSC supernatants and recombinant cytokines. Cell surface markers were assessed by flow cytometry, secreted cytokines measured using ELISA, T cell proliferation by CFSE, Treg induction by flow cytometry and phagocytosis by fluorescent bead uptake. Western blot assays were used to assess activation of the p-STAT1 and p-STAT3 pathways. The p-STAT3 pathway was inhibited with the small molecule WP1066 and siRNA. Results: The gCSCs produce macrophage inhibitory cytokine (MIC-1) (22.3 - 1056.2 pg/106 cells/24 hours), macrophage inhibitory factor (MIF) (69.1 - 1463.3 pg/106 cells/24 hours), and TGF-β1 (24 - 73.8 pg/106 cells/24 hours) but not IL-23. The MDMs exposed to the gCSC supernatants increased expression of CD80 and B7-H1, and had significantly potentiated secretion of TGF-β1 (106.3 - 3001.8 pg/106 cells/24 hours) and IL-23 (176.8 - 5650.6 pg/106 cells/24 hours). The induced TGF-β1 and IL-23 correlated with inhibition of T cell proliferation and the induction of Tregs. MDM phagocytosis was markedly inhibited by the gCSC supernatants, and physiological amounts of recombinant MIC-1 and MIF. Similar findings were obtained with the human microglia. The gCSC supernatants up-regulated p-STAT3 in the MDMs; however the M2 phenotype could be reversed when p-STAT3 was blocked in either the gCSCs or the MDMs. Conclusions: The gCSCs secrete factors such as MIC-1 and MIF that can induce immune suppression in macrophages characterized by altered expression of cell surface markers, increased secretion of TGF-β1 and IL-23 with the associated ability to inhibit T cell proliferation and induce Tregs, and impaired phagocytosis. The M2 phenotype is reversed with p-STAT3 blockade. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5322.