Anai N. Kothari

Novel clinical therapeutics targeting the epithelial to mesenchymal transition

The epithelial to mesenchymal transition (EMT) is implicated in many processes, ranging from tissue and organogenesis to cancer and metastatic spread. Understanding the key regulatory mechanisms and mediators within this process offers the opportunity to develop novel therapeutics with broad clinical applicability. To date, several components of EMT already are targeted using pharmacologic agents in fibrosis and cancer. As our knowledge of EMT continues to grow, the potential for novel therapeutics will also increase. This review focuses on the role of EMT both as a necessary part of development and a key player in disease progression, specifically the similarity in pathways used during both processes as targets for drug development. Also, the key role of the tumor microenvironment with EMT is outlined, focusing on both co-factors and cell types with the ability to modulate the progression of EMT in cancer and metastatic disease. Lastly, we discuss the current status of clinical therapies both in development and those progressed to clinical trial specifically targeting pathologic EMTs including small molecule inhibitors, non-coding RNAs, exogenous co-factors, and adjunctive therapies to current chemotherapeutics.

Osteopontin—A Master Regulator of Epithelial-Mesenchymal Transition

Osteopontin (OPN) plays an important functional role in both physiologic and pathologic states. OPN is implicated in the progression of fibrosis, cancer, and metastatic disease in several organ systems. The epithelial-mesenchymal transition (EMT), first described in embryology, is increasingly being recognized as a significant contributor to fibrotic phenotypes and tumor progression. Several well-established transcription factors regulate EMT and are conserved across tissue types and organ systems, including TWIST, zinc finger E-box-binding homeobox (ZEB), and SNAIL-family members. Recent literature points to an important relationship between OPN and EMT, implicating OPN as a key regulatory component of EMT programs. In this review, OPN’s interplay with traditional EMT activators, both directly and indirectly, will be discussed. Also, OPN’s ability to restructure the tissue and tumor microenvironment to indirectly modify EMT will be reviewed. Together, these diverse pathways demonstrate that OPN is able to modulate EMT and provide new targets for directing therapeutics.

Alcohol Inhibits Osteopontin Dependent Transforming Growth Factor-β1 Expression in Human Mesenchymal Stem Cells

Background: Alcohol (EtOH) exposure has detrimental effects on fracture healing. Results: EtOH inhibits TGF-β1 protein expression via interference with the transcription factor myeloid zinc finger 1. Conclusion: EtOH-induced deficient fracture healing may be related to reduced TGF-β1. Significance: Further understanding of the mechanisms responsible for the effects of EtOH are crucial for the development of interventions aimed at averting morbidities related to EtOH consumption. Alcohol (EtOH) intoxication is a risk factor for increased morbidity and mortality with traumatic injuries, in part through inhibition of bone fracture healing. Animal models have shown that EtOH decreases fracture callus volume, diameter, and biomechanical strength. Transforming growth factor β1 (TGF-β1) and osteopontin (OPN) play important roles in bone remodeling and fracture healing. Mesenchymal stem cells (MSC) reside in bone and are recruited to fracture sites for the healing process. Resident MSC are critical for fracture healing and function as a source of TGF-β1 induced by local OPN, which acts through the transcription factor myeloid zinc finger 1 (MZF1). The molecular mechanisms responsible for the effect of EtOH on fracture healing are still incompletely understood, and this study investigated the role of EtOH in affecting OPN-dependent TGF-β1 expression in MSC. We have demonstrated that EtOH inhibits OPN-induced TGF-β1 protein expression, decreases MZF1-dependent TGF-β1 transcription and MZF1 transcription, and blocks OPN-induced MZF1 phosphorylation. We also found that PKA signaling enhances OPN-induced TGF-β1 expression. Last, we showed that EtOH exposure reduces the TGF-β1 protein levels in mouse fracture callus. We conclude that EtOH acts in a novel mechanism by interfering directly with the OPN-MZF1-TGF-β1 signaling pathway in MSC.

Postoperative Atrial Fibrillation Predicts Long-Term Cardiovascular Events after Radical Cystectomy

PURPOSE Postoperative atrial fibrillation after radical cystectomy occurs in 2% to 8% of cases. Recent evidence suggests that transient postoperative atrial fibrillation leads to future cardiovascular events. The long-term cardiovascular implications of postoperative atrial fibrillation in patients undergoing radical cystectomy are largely unknown. MATERIALS AND METHODS The Healthcare Cost and Utilization Project State Inpatient Databases for California and Florida were used to identify patients who underwent radical cystectomy between 2007 and 2010. After excluding patients with a history of atrial fibrillation, coronary artery disease and/or stroke, patients were matched using propensity scoring on age, race, insurance status and preexisting comorbidities. Adjusted Kaplan-Meier time-to-event analysis and Cox proportional hazards models were used to assess the effect of postoperative atrial fibrillation on cardiovascular events (acute myocardial infarction and stroke) during postoperative year 1. RESULTS Radical cystectomy was performed in 4,345 patients who met the study inclusion criteria, of whom 210 (4.8%) had postoperative atrial fibrillation. There was a significantly higher cumulative incidence of cardiovascular events during the first postoperative year in patients in whom postoperative atrial fibrillation developed (24.8% vs 10.9%, adjusted log rank p=0.007). Cox proportional hazards regression demonstrated an increased risk of cardiovascular events in patients with postoperative atrial fibrillation (HR 10, p=0.02). CONCLUSIONS Our results demonstrate that patients undergoing radical cystectomy in whom transient postoperative atrial fibrillation develops are at significantly increased risk for cardiovascular events in the first postoperative year. Physicians should be vigilant in assessing postoperative atrial fibrillation, even when transient, and establish appropriate followup given the increased risk of cardiovascular morbidity.

Green tea component epigallocatechin-3-gallate decreases expression of osteopontin via a decrease in mRNA half-life in cell lines of metastatic hepatocellular carcinoma

INTRODUCTION Osteopontin (OPN) mediates metastasis and invasion of hepatocellular carcinoma (HCC). Epigallocatechin-3-gallate (EGCG), found in green tea, suppresses HCC tumor growth in vitro. We sought to investigate the role of EGCG in modulating OPN in cell lines of metastatic HCC. METHODS Experimental HCC cell lines included HepG2 and MHCC-97H HCC cells, which express high levels of OPN, and the Hep3B cells, which express lesser levels of OPN. Cells were treated with EGCG (0.02-20 μg/mL) before measurement of OPN with enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. Scratch assay measured cell migration. Binding of the OPN promoter to RNA pol II was evaluated by the use of Chromatin-IP assay after EGCG treatment. Transcriptional regulation of OPN was investigated with luciferase reporter plasmids containing various deletion fragments of the human OPN promoter. Measurement of the half-life of OPN mRNA was conducted using actinomycin D. RESULTS Treatment of MHCC-97H and HepG2 cells with 2 μg/mL and 20 μg/mL EGCG caused a ∼6-fold and ∼90-fold decrease in secreted protein levels of OPN (All P < .001). OPN mRNA was decreased with EGCG concentrations of 0.2-20 μg/ml (All P < .001). The 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (ie, MTT) assay revealed that differences in OPN expression were not due to viability of the HCC cell lines. Promoter assay and chromatin immunoprecipitation analysis revealed no effect of EGCG on the transcriptional regulation of OPN. Posttranscriptionally, EGCG decreased the half-life of OPN mRNA from 16.8 hours (95% confidence interval 9.0-125.1) to 2.5 hours (95% confidence interval 2.1-3.2) (P < .001). Migration was decreased in EGCG treated cells at 24 hours (8.0 ± 2.4% vs 21.2 ± 10.8%, P < .01) and at 48 hours (13.2 ± 3.6% vs 53.5 ± 19.8%, P < .001). CONCLUSION We provide evidence that EGCG decreases OPN mRNA and secreted OPN protein levels by decreasing the half-life of OPN mRNA in MHCC-97H cells. The translatability of EGCG for patients with HCC is promising, because EGCG is an inexpensive, easily accessible chemical with an extensive history of safety.

Epigallocatechin-3-Gallate Upregulates miR-221 to Inhibit Osteopontin-Dependent Hepatic Fibrosis.

Osteopontin (OPN) promotes hepatic fibrosis, and developing therapies targeting OPN expression in settings of hepatic injury holds promise. The polyphenol epigallocatechin-3-gallate (EGCG), found in high concentrations in green tea, downregulates OPN expression through OPN mRNA degradation, but the mechanism is unknown. Previous work has shown that microRNAs can decrease OPN mRNA levels, and other studies have shown that EGCG modulates the expression of multiple microRNAs. In our study, we first demonstrated that OPN induces hepatic stellate cells to transform into an activated state. We then identified three microRNAs which target OPN mRNA: miR-181a, miR-10b, and miR-221. In vitro results show that EGCG upregulates all three microRNAs, and all three microRNAs are capable of down regulating OPN mRNA when administered alone. Interestingly, only miR-221 is necessary for EGCG-mediated OPN mRNA degradation and miR-221 inhibition reduces the effects of EGCG on cell function. In vivo experiments show that thioacetamide (TAA)-induced cell cytotoxicity upregulates OPN expression; treatment with EGCG blocks the effects of TAA. Furthermore, chronic treatment of EGCG in vivo upregulates all three microRNAs equally, suggesting that in more chronic treatment all three microRNAs are involved in modulating OPN expression. We conclude that in in vitro and in vivo models of TAA-induced hepatic fibrosis, EGCG inhibits OPN-dependent injury and fibrosis. EGCG works primarily by upregulating miR-221 to accelerate OPN degradation. EGCG may therefore have utility as a protective agent in settings of liver injury.

Take the Volume Pledge may result in disparity in access to care.

Background. “Take the Volume Pledge” proposes restricting pancreatectomies to hospitals that perform ≥20 per year. Our purpose was to identify those factors that characterize patients at risk for loss of access to pancreatic cancer care with enforcement of volume standards. Methods. Using the Healthcare Cost and Utilization Project State Inpatient Database from Florida, we identified patients who underwent pancreatectomy for pancreatic malignancy from 2007–2011. American Hospital Association and United States Census Bureau data were linked to patient‐level data. High‐volume hospitals were defined as performing ≥20 pancreatic resections per year. Univariable and multivariable statistics compared patient characteristics and utilization of high‐volume hospitals. Classification and Regression Tree modeling was used to predict patients at risk for losing access to care. Results. Our study included 1,663 patients. Five high‐volume hospitals were identified, and they treated 1,056 (63.5%) patients. Patients residing far from high‐volume hospitals, in areas with the highest population density, non‐Caucasian ethnicity, and greater income had decreased odds of obtaining care at high‐volume hospitals. Using these factors, we developed a Classification and Regression Tree–based predictive tool to identify these patients. Conclusion. Implementation of “Take the Volume Pledge” is an important step toward improving pancreatectomy outcomes; however, policymakers must consider the potential impact on limiting access and possible health disparities that may arise.

Incidence of Adverse Contrast Reaction Following Nonintravenous Urinary Tract Imaging

Adverse reactions (ARs) to intravenous (IV) radiographic contrast range from mild urticaria to life-threatening anaphylaxis. Intraluminal contrast dye is routinely used in the urinary tract with a minimal perceived risk of AR. We used the Healthcare Cost and Utilization Project State Inpatient Databases for California and Florida from 2007 to 2011 to identify patients who received urinary tract contrast dye for retrograde pyelography, percutaneous pyelography, retrograde/other cystogram, and ileal conduitogram. After excluding patients who had received IV contrast for other radiologic studies, ARs to contrast were identified by a composite end point of diagnoses not present on admission including shock, anaphylaxis, iatrogenic hypotension, urticaria, angioedema, laryngospasm, laryngeal edema, and/or a new diagnosis of contrast reaction. Overall, 76 174 patients were included who had undergone non-IV urinary tract imaging, 367 (0.48%) of whom developed an AR. On multivariate analysis, receipt of contrast in the lower urinary tract (odds ratio [OR]: 1.8; p=0.04) or upper urinary tract by retrograde pyelography (OR: 1.6; p=0.04) or antegrade pyelography (OR: 2.0; p=0.007) increased the risk of AR compared with control patients. The use of contrast dye in the urinary tract is associated with a low, but present risk of AR. PATIENT SUMMARY We looked at patients who underwent a urologic procedure using radiographic contrast media in the urinary tract. Although adverse reactions (ARs) may occur with the use of contrast media in the urinary tract, these reactions are experienced by a minority of patients (approximately 1 in 200). In addition, we found that an allergy to intravenous contrast does not increase a patients risk of an AR to contrast within the urinary tract.

Right place at the right time impacts outcomes for acute intestinal obstruction.

BACKGROUND The purpose of this study was to measure how the duration of nonoperative intervention for intestinal obstruction impacted patient outcomes and whether hospital characteristics influenced the timing of operative intervention. METHODS The State Inpatient Database (Florida) of the Health Care Utilization Project and the Annual Survey database of the American Hospital Association were linked from 2006 to 2011. Included were patients ≥18 years of age with a primary diagnosis of intestinal obstruction. Patient factors included age, sex, socioeconomic factors, and comorbid conditions. RESULTS A total of 116,195 patients met our inclusion criteria, and 43,079 underwent operative intervention (37.1%). Patients who required operative correction of the intestinal obstruction after the fifth day of hospitalization, compared with patients who underwent an operation on the day of admission, had increases in mortality (6.1% vs 1.8%, P < .001), complication rates (15.4% vs 4.0%, P < .001), and postoperative hospital stay (9 vs 5 days, P < .001). Patients cared for at a large teaching facility (with surgery residents) had increased odds of early operative intervention by 23% (odds ratio 1.23, [1.20-1.28]), whereas patients at low-volume hospitals had decreased odds of early intervention (odds ratio 0.88, [0.73-0.91]). CONCLUSION Initial nonoperative treatment in patients with uncomplicated intestinal obstruction is an important strategy, but the odds of having an adverse event increase as intestinal obstruction is delayed. Importantly, the presence of surgery residents and increasing bed size are hospital characteristics associated with earlier operative intervention, suggesting a quality benefit for care at large teaching hospitals.

Impact of Post-Hospital Syndrome on Outcomes Following Elective, Ambulatory Surgery.

Objective: The aim of this study was to investigate whether post-hospital syndrome (PHS) places patients undergoing elective hernia repair at increased risk for adverse postoperative events. Summary of Background Data: PHS is a transient period of health vulnerability following inpatient hospitalization for acute illness. PHS has been well studied in nonsurgical populations, but its effect on surgical outcomes is unclear. Methods: State-specific datasets for California in 2011 available through the Healthcare Cost and Utilization Project (HCUP) were linked. Patients older than 18 years who underwent elective hernia repair were included. The primary exposure variable was PHS, defined as any inpatient admission within 90 days of an elective hernia repair performed in an ambulatory surgery center. The primary outcome was an adverse event, defined as any unplanned emergency department visit or inpatient admission within 30 days postoperatively. Mixed-effects logistic models were used for multivariable analyses. Results: A total of 57,988 patients met inclusion criteria. The 30-day risk-adjusted adverse event rate was significantly higher for PHS patients versus non-PHS patients (11.8% vs 5.8%, P < 0.001). PHS patients were more likely than non-PHS patients to experience postoperative complications (odds ratio 2.2, 95% confidence interval 1.6–3.0). Adverse events attributable to PHS cost an additional