UHRF1/UBE2L6/UBR4-mediated ubiquitination regulates EZH2 abundance and thereby melanocytic differentiation phenotypes in melanoma.

Cellular heterogeneity in cancer is linked to disease progression and therapy response, although mechanisms regulating distinct cellular states within tumors are not well understood. We identified melanin pigment content as a major source of cellular heterogeneity in melanoma and compared RNAseq data from high-pigmented (HPCs) and low-pigmented melanoma cells (LPCs), suggesting EZH2 as a master regulator of these states. EZH2 protein was found to be upregulated in LPCs and inversely correlated with melanin deposition in pigmented patient melanomas. Surprisingly, conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, had no effect on LPC survival, clonogenicity and pigmentation, despite fully inhibiting methyltransferase activity. In contrast, EZH2 silencing by siRNA or degradation by DZNep or MS1943 inhibited growth of LPCs and induced HPCs. As the proteasomal inhibitor MG132 induced EZH2 protein in HPCs, we evaluated ubiquitin pathway proteins in HPC vs LPCs. Biochemical assays and animal studies demonstrated that in LPCs, the E2-conjugating enzyme UBE2L6 depletes EZH2 protein in cooperation with UBR4, an E3 ligase, via ubiquitination at EZH2's K381 residue, and is downregulated in LPCs by UHRF1-mediated CpG methylation. Targeting UHRF1/UBE2L6/UBR4-mediated regulation of EZH2 offers potential for modulating the activity of this oncoprotein in contexts in which conventional EZH2 methyltransferase inhibitors are ineffective.

The EUS molecular evaluation of pancreatic cancer: A prospective multicenter cohort trial.

BACKGROUND AND OBJECTIVES: Patients with locally advanced or metastatic pancreatic ductal adenocarcinoma (A-PDAC) are not candidates for surgical resection and are often offered palliative chemotherapy. The ready availability of a safe and effective tumor sampling technique to provide material for both diagnosis and comprehensive genetic profiling is critical for informing precision medicine in A-PDAC, thus potentially increasing survival. The aim of this study is to examine the feasibility and benefits of routine comprehensive genomic profiling (CGP) of A-PDAC using EUS-FNA material. METHODS: This is a prospective cohort study to test the clinical utility of fresh frozen or archival EUS-FNA samples in providing genetic material for CGP. The results of the CGP will be reviewed at a molecular tumor board. The proportion of participants that have a change in their treatment recommendations based on their individual genomic profiling will be assessed. Correlations between CGP and stage, prognosis, response to treatment and overall survival will also be investigated. This study will open to recruitment in 2020, with a target accrual of 150 A-PDAC patients within 36 months, with a 2-year follow-up. It is expected that the majority of participants will be those who have already consented for their tissue to be biobanked in the Victorian Pancreatic Cancer Biobank at the time of diagnostic EUS-FNA. Patients without archival or biobanked material that is suitable for CGP may be offered a EUS-FNA procedure for the purposes of obtaining fresh frozen material. DISCUSSION: This trial is expected to provide crucial data regarding the feasibility of routine CGP of A-PDAC using EUS-FNA material. It will also provide important information about the impact of this methodology on patients' survival.

Potentiation of chemotherapeutics by bromelain and N-acetylcysteine: sequential and combination therapy of gastrointestinal cancer cells.

Intraperitoneal chemotherapy together with cytoreductive surgery is the standard of care for a number of peritoneal surface malignancies. However, this approach fails to maintain the complete response and disease recurs due to microscopic residual disease. Although safer than systemic chemotherapy regimens, locoregional treatment with chemotherapeutics can induce toxicity which is a major concern affecting the patient's treatment protocol and outcome. For an enhanced treatment efficacy, efforts should be made to maximize cytotoxic effects of chemotherapeutic agents on tumor cells while minimizing their toxic effects on host cells. Bromelain and N-acetylcysteine are two natural agents with good safety profiles shown to have anti-cancer effects. However, their interaction with chemotherapeutics is unknown. In this study, we investigated if these agents have the potential to sensitize in vitro gastrointestinal cancer models to cisplatin, paclitaxel, 5-fluorouracil, and vincristine. The drug-drug interaction was also analyzed. Our findings suggest that combination of bromelain and N-acetylcysteine with chemotherapeutic agents could give rise to an improved chemotherapeutic index in therapeutic approaches to peritoneal surface malignancies of gastrointestinal origin so that maximum benefits could result from less toxic and more patient-friendly doses. This represents a potentially efficacious strategy for the enhancement of microscopic cytoreduction and is a promising area for future research.

Depletion of mucin in mucin-producing human gastrointestinal carcinoma: Results from in vitro and in vivo studies with bromelain and N-acetylcysteine.

Aberrant expression of membrane-associated and secreted mucins, as evident in epithelial tumors, is known to facilitate tumor growth, progression and metastasis, and to provide protection against adverse growth conditions, chemotherapy and immune surveillance. Emerging evidence provides support for the oncogenic role of MUC1 in gastrointestinal carcinomas and relates its expression to an invasive phenotype. Similarly, mucinous differentiation of gastrointestinal tumors, in particular increased or de novo expression of MUC2 and/or MUC5AC, is widely believed to imply an adverse clinicopathological feature. Through formation of viscous gels, too, MUC2 and MUC5AC significantly contribute to the biology and pathogenesis of mucin-secreting gastrointestinal tumors. Here, we investigated the mucin-depleting effects of bromelain (BR) and N-acetylcysteine (NAC), in nine different regimens as single or combination therapy, in in vitro (MKN45, KATOIII and LS174T cell lines) and in vivo (female nude mice bearing intraperitoneal MKN45 and LS174T) settings. The inhibitory effects of the treatment on cancer cell growth and proliferation were also evaluated in vivo. Our results suggest that a combination of BR and NAC with dual effects on growth and mucin products of mucin-expressing tumor cells is a promising candidate towards the development of novel approaches to gastrointestinal malignancies with the involvement of mucin pathology. This capability supports the use of this combination formulation in locoregional approaches for reducing the adverse effects of the aberrantly secreted gel-forming mucins, as in pseudomyxoma peritonei and similar pathologies with ectopic production of mucin.

Sprouty2 protein in prediction of post-treatment ascites in epithelial ovarian cancer treated with adjuvant carbotaxol chemotherapy.

Ascites development and resistance to chemotherapy with carbotaxol are common clinical problems in epithelial ovarian cancer, partly due to the activation of MAPK/ERK signaling. Sprouty proteins are negative modulators of MAPK/ERK pathway, but their role in predicting resistance to carbotaxol chemotherapy and ascites development is unknown. In this study, we evaluated the expression of Sprouty protein isoforms by immunohistochemistry. The associations between the Sprouty expression and the clinicopathological features, including chemoresistance and the presence of ascites, were then explored. We found that the decreased expression of Spry2 was correlated with the post-treatment development of ascites and represented an independent predictor of this condition in carbotaxol-treated patients. However, no association was observed between the Sprouty expression and chemoresistance. In conclusion, our results suggest that Spry2 may be useful for patient follow-up and monitoring as it predicts the development of ascites in epithelial ovarian cancer cases treated with carbotaxol.

Intratumoral interleukin-6 predicts ascites formation in patients with epithelial ovarian cancer: A potential tool for close monitoring.

BACKGROUND: The implication of IL-6 in the pathogenesis of epithelial ovarian cancer (EOC) is well documented. Accordingly, the clinicopathological significance of this cytokine in patients' ascites fluid or serum has largely been investigated. Since the main source of IL-6 secreted into the biological fluids is the tumor tissue, this study was designed to investigate the status and possible clinical relevance of the IL-6 expression in an array of EOC tissue specimens. METHODS: Tissue samples obtained from ninety-eight consecutive patients with EOC were studied using immunohistochemistry. Clinicopathological characteristics and treatment related factors were collected from patient files. The relationship between the expression of the protein of interest and the study endpoints of disease-free survival (DFS) and overall survival (OS) were analyzed using the Kaplan-Meier method. For evaluating the predictive value of IL-6, logistic regression and cox proportional hazards models were employed. RESULTS: An upregulation of IL-6 expression was observed in EOC tissues as compared with the normal samples (p < 0.0001). As regards the clinical relevance, IL-6 failed to predict OS, DFS and response to the platinum-based chemotherapy in EOC patients. In multivariate analysis, however, IL-6 was identified as an independent predictive factor for the development of post-treatment ascites (p:0.033). CONCLUSIONS: Having the capability to predict the ascites formation, IL-6 might serve as a biomarker and a useful tool in EOC for monitoring purposes. IL-6 targeting for the prevention of the ascites development is a potential avenue for further investigation.

Vascular endothelial growth factor expression correlates with serum CA125 and represents a useful tool in prediction of refractoriness to platinum-based chemotherapy and ascites formation in epithelial ovarian cancer.

There is an increasing need for the identification of novel biological markers and potential therapeutic targets in epithelial ovarian cancer (EOC). Given the critical role of growth factors in the biology of EOC, we aimed in the present study to evaluate the intratumoral expressions of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) proteins and their clinical relevance in a cohort of 100 patients with EOC. All patients received platinum-based chemotherapy after surgery. A comparative immunohistochemical study of normal ovarian and EOC tissues showed that both growth factors were expressed at higher levels in tumor samples. In our statistical analysis, while no association existed between the FGF expression status and the clinicopathological characteristics of patients, intratumoral VEGF was identified as a potential biomarker for the prediction of ascites formation. In addition, the expression status of VEGF appeared to independently predict overall survival and response to chemotherapy. Furthermore, a direct association was demonstrated between the pre-treatment VEGF expression and serum CA125 after three cycles of chemotherapy. In sum, we report for the first time to our knowledge the correlation between intratumoral VEGF and serum CA125 in EOC. Our data also shows the prognostic value of VEGF expression in EOC. These results suggest the potential value of intratumoral VEGF in patient stratification. Dual inhibition of VEGF and CA125 might bring about a better outcome for patients with EOC.

Sprouty 1 predicts prognosis in human epithelial ovarian cancer.

Sprouty proteins are evolutionary-conserved modulators of receptor tyrosine kinase (RTK) signaling. We have previously reported inverse correlation of the Sprouty 1 (Spry1) protein expression with ovarian cancer cell proliferation, migration, invasion and survival. In the present study, the expression status of Spry1 protein and its clinical relevance in patients with epithelial ovarian cancer were explored. Matched tumor and normal tissue samples from 100 patients with epithelial ovarian cancer were immunohistochemically stained for Spry1. Expression of ERK, p-ERK, Ki67, FGF-2, VEGF and IL-6 and their correlation with Spry1 were also evaluated. In addition, correlation between Spry1 and clinicopathological characteristics and predictive significance of Spry1 for overall survival (OS) and disease-free survival (DFS) were analysed. Our data indicated that Spry1 was significantly downregulated in tumor tissues (p=0.004). Spry1 showed significant inverse correlation with p-ERK/ERK (p=0.045), Ki67 (p=0.010), disease stage (p=0.029), tumor grade (p=0.037), recurrence (p=0.001) and lymphovascular invasion (p=0.042). It was revealed that Spry1 low-expressing patients had significantly poorer OS (p=0.010) and DFS (p=0.012) than those with high expression of Spry1. Multivariate analysis showed that high Spry1 (p=0.030), low stage (p=0.048) and no residual tumor (p=0.007) were independent prognostic factors for a better OS, among which high Spry1 (p=0.035) and low stage (p=0.035) remained as independent predictors of DFS, too. We also found that the expression of Spry1 significantly correlates with the expression of Spry2 (p<0.001), but not that of Spry4. In conclusion, we report for the first time to our knowledge that Spry1 protein is downregulated in human epithelial ovarian cancer. Spry1 expression significantly impacts tumor behavior and shows predictive value as an independent prognostic factor for survival and recurrence.

Sprouty 2 protein, but not Sprouty 4, is an independent prognostic biomarker for human epithelial ovarian cancer.

Sprouty proteins are evolutionary-conserved modulators of receptor tyrosine kinase signaling, deregulation of which has been implicated in the pathophysiology of cancer. In the present study, the expression status of Spry2 and Spry4 proteins and its clinical relevance in human epithelial ovarian cancer (EOC) were investigated retrospectively. We examined the immunohistochemical expression of Spry2 and Spry4 in matched tumor and normal tissue samples from 99 patients. The expression of ERK, p-ERK, Ki67, fibroblast growth factor-2, vascular endothelial growth factor and interleukin-6 and their correlation with Sprouty homologs were also evaluated. Moreover, the correlation between Spry2 and Spry4 and the clinicopathological characteristics were analyzed along with their predictive value for overall survival (OS) and disease-free survival (DFS). Our data indicated significant downregulation of Spry2 and Spry4 in tumor tissues (p < 0.0001). A significant inverse correlation was evident between Spry2 and p-ERK/ERK (p = 0.048), Ki67 (p = 0.011), disease stage (p = 0.013), tumor grade (p = 0.003), recurrence (p < 0.001) and post-treatment ascites (p = 0.001), individually. It was found that Spry2 low-expressing patients had significantly poorer OS (p = 0.002) and DFS (p = 0.004) than those with high expression of Spry2. Multivariate analysis showed that high Spry2 (p = 0.018), low stage (p = 0.049) and no residual tumor (p =0.006) were independent prognostic factors for a better OS. With regard to DFS, high Spry2 (p = 0.044) and low stage (p = 0.046) remained as independent predictors. In conclusion, we report for the first time significant downregulation of Spry2 and Spry4 proteins in human EOC. Spry2 expression was revealed to significantly impact tumor behavior with predictive value as an independent prognostic factor for survival and recurrence.

Bromelain and N-acetylcysteine inhibit proliferation and survival of gastrointestinal cancer cells in vitro: significance of combination therapy.

BACKGROUND: Bromelain and N-acetylcysteine are two natural, sulfhydryl-containing compounds with good safety profiles which have been investigated for their benefits and application in health and disease for more than fifty years. As such, the potential values of these agents in cancer therapy have been variably reported in the literature. In the present study, the efficacy of bromelain and N-acetylcysteine in single agent and combination treatment of human gastrointestinal carcinoma cells was evaluated in vitro and the underlying mechanisms of effect were explored. METHODS: The growth-inhibitory effects of bromelain and N-acetylcysteine, on their own and in combination, on a panel of human gastrointestinal carcinoma cell lines, including MKN45, KATO-III, HT29-5F12, HT29-5M21 and LS174T, were assessed by sulforhodamine B assay. Moreover, the influence of the treatment on the expression of a range of proteins involved in the regulation of cell cycle and survival was investigated by Western blot. The presence of apoptosis was also examined by TUNEL assay. RESULTS: Bromelain and N-acetylcysteine significantly inhibited cell proliferation, more potently in combination therapy. Drug-drug interaction in combination therapy was found to be predominantly synergistic or additive. Mechanistically, apoptotic bodies were detected in treated cells by TUNEL assay. Furthermore, Western blot analysis revealed diminution of cyclins A, B and D, the emergence of immunoreactive subunits of caspase-3, caspase-7, caspase-8 and cleaved PARP, withering or cleavage of procaspase-9, overexpression of cytochrome c, reduced expression of anti-apoptotic Bcl-2 and pro-survival phospho-Akt, the emergence of the autophagosomal marker LC3-II and deregulation of other autophagy-related proteins, including Atg3, Atg5, Atg7, Atg12 and Beclin 1. These results were more prominent in combination therapy. CONCLUSION: We report for the first time to our knowledge the growth-inhibitory and cytotoxic effects of bromelain and N-acetylcysteine, in particular in combination, on a panel of gastrointestinal cancer cell lines with different phenotypes and characteristics. These effects apparently resulted from cell cycle arrest, apoptosis and autophagy. Towards the development of novel strategies for the enhancement of microscopic cytoreduction, our results lay the basis for further evaluation of this formulation in locoregional approaches to peritoneal surface malignancies and carcinomatosis.

Secreted mucins in pseudomyxoma peritonei: pathophysiological significance and potential therapeutic prospects.

Pseudomyxoma peritonei (PMP, ORPHA26790) is a clinical syndrome characterized by progressive dissemination of mucinous tumors and mucinous ascites in the abdomen and pelvis. PMP is a rare disease with an estimated incidence of 1-2 out of a million. Clinically, PMP usually presents with a variety of unspecific signs and symptoms, including abdominal pain and distention, ascites or even bowel obstruction. It is also diagnosed incidentally at surgical or non-surgical investigations of the abdominopelvic viscera. PMP is a neoplastic disease originating from a primary mucinous tumor of the appendix with a distinctive pattern of the peritoneal spread. Computed tomography and histopathology are the most reliable diagnostic modalities. The differential diagnosis of the disease includes secondary peritoneal carcinomatoses and some rare peritoneal conditions. Optimal elimination of mucin and the mucin-secreting tumor comprises the current standard of care for PMP offered in specialized centers as visceral resections and peritonectomy combined with intraperitoneal chemotherapy. This multidisciplinary approach has reportedly provided a median survival rate of 16.3 years, a median progression-free survival rate of 8.2 years and 10- and 15-year survival rates of 63% and 59%, respectively. Despite its indolent, bland nature as a neoplasm, PMP is a debilitating condition that severely impacts quality of life. It tends to be diagnosed at advanced stages and frequently recurs after treatment. Being ignored in research, however, PMP remains a challenging, enigmatic entity. Clinicopathological features of the PMP syndrome and its morbid complications closely correspond with the multifocal distribution of the secreted mucin collections and mucin-secreting implants. Novel strategies are thus required to facilitate macroscopic, as well as microscopic, elimination of mucin and its source as the key components of the disease. In this regard, MUC2, MUC5AC and MUC5B have been found as the secreted mucins of relevance in PMP. Development of mucin-targeted therapies could be a promising avenue for future research which is addressed in this article.

The expression of the Sprouty 1 protein inversely correlates with growth, proliferation, migration and invasion of ovarian cancer cells.

BACKGROUND: Our recent study on a panel of human ovarian cancer cells revealed that SKOV-3 cells barely express the Sprouty isoform 1 (Spry1) while 1A9 cells maintain it at a level similar to normal ovarian cells. Here we investigated the functional outcomes of induced alterations in the expression of Spry1 in the two cell lines in vitro. METHODS: Using the Spry1 specific plasmid and siRNA, the expression of Spry1 was induced and conversely silenced in SKOV-3 and 1A9 cells, respectively. The functional outcome was investigated by means of proliferation, MTT, scratch-wound, migration and invasion assays and selection of the stable clones. Mechanism of the effect was explored by Western blot. RESULTS: In the Spry1-transfected SKOV-3 cells, a significant reduction in growth and proliferation was evident. Stable clones of the Spry1-transfected SKOV-3 were almost undetectable after day 14. The number of migrated and invaded cells and the percentage of the scratch closure were significantly lower in the Spry1-transfected group. Spry1 silencing in 1A9 cells, on the other hand, led to a significant increase in cell growth and proliferation. The number of migrated and invaded cells and the percentage of the scratch closure significantly increased in Spry1-silenced 1A9 group. Mechanistically, overexpression of Bax, activation of caspases 3, 7, 8 and 9, cleavage of PARP and attenuation of Bcl-2 and Bcl-xl were observed along with reduced activation of Erk and Akt and increased amount and activity of PTEN in the Spry1-transfected SKOV-3 cells. CONCLUSIONS: Here, we report the inverse correlation between the expression of Spry1 and growth, proliferation, invasion and migration of ovarian cancer cells.

The developing story of Sprouty and cancer.

Sprouty proteins are evolutionarily conserved modulators of MAPK/ERK pathway. Through interacting with an increasing number of effectors, mediators, and regulators with ultimate influence on multiple targets within or beyond ERK, Sprouty orchestrates a complex, multilayered regulatory system and mediates a crosstalk among different signaling pathways for a coordinated cellular response. As such, Sprouty has been implicated in various developmental and physiological processes. Evidence shows that ERK is aberrantly activated in malignant conditions. Accordingly, Sprouty deregulation has been reported in different cancer types and shown to impact cancer development, progression, and metastasis. In this article, we have tried to provide an overview of the current knowledge about the Sprouty physiology and its regulatory functions in health, as well as an updated review of the Sprouty status in cancer. Putative implications of Sprouty in cancer biology, their clinical relevance, and their proposed applications are also revisited. As a developing story, however, role of Sprouty in cancer remains to be further elucidated.

Cytotoxic effects of bromelain in human gastrointestinal carcinoma cell lines (MKN45, KATO-III, HT29-5F12, and HT29-5M21).

BACKGROUND: Bromelain is a pineapple stem extract with a variety of therapeutic benefits arising from interaction with a number of different biological processes. Several preclinical studies and anecdotal clinical observations have reported the anticancer properties of bromelain. In the present study, we investigated the cytotoxic effects of bromelain in four human cancer cell lines of gastrointestinal origin and the mechanisms involved. METHODS: The gastric carcinoma cell lines (KATO-III and MKN45) and two chemoresistant subpopulations of the HT29 colon adenocarcinoma cell line (HT29-5M21 and HT29-5F12) were treated with a range of concentrations of bromelain, as well as with cisplatin as a positive control. The effect of bromelain on the growth and proliferation of cancer cells was determined using a sulforhodamine B assay after 72 hours of treatment. Expression of apoptosis-associated proteins in MKN45 cells treated with bromelain was analyzed by Western blotting. RESULTS: Data from our sulforhodamine B assay showed that bromelain inhibited proliferation of HT29-5F12, HT29-5M21, MKN45, and KATO-III cells, with respective half maximal inhibitory concentration values of 29, 34, 94, and 142 µg/mL. Analyzing the expression of proapoptotic and antiapoptotic proteins in bromelain-treated MKN45 cells, we observed activation of the caspase system, cleavage of PARP and p53, overexpression of cytochrome C, attenuation of phospho-Akt and Bcl2, and removal of MUC1. Apart from the caspase-dependent apoptosis observed, emergence of cleaved p53 supports a direct, extranuclear apoptotic function of p53. Moreover, interrupted Akt signaling and attenuation of Bcl2 and MUC1 oncoproteins suggest impaired survival of cancer cells. CONCLUSION: Our findings collectively indicate that bromelain exerts cytotoxic effects in a panel of human gastric and colon carcinoma cells. Our study of MKN45 cells implicated different mechanisms in bromelain-induced cell death. While promoting apoptosis with involvement of the caspase system and extranuclear p53, bromelain also appears to impair cancer cell survival by blocking the Akt pathway and attenuating Bcl2 and MUC1 oncoproteins.

Utility of vascular endothelial growth factor inhibitors in the treatment of ovarian cancer: from concept to application.

Despite recent advances in the management of ovarian cancer, it remains the most lethal gynecologic malignancy. Vascular endothelial growth factor (VEGF) has been shown to play a pivotal role in the progression of ovarian cancer leading to the eventual development of malignant ascites. On this basis, agents rendering VEGF ineffective by neutralizing VEGF (bevacizumab), blocking its receptors (aflibercept), or interfering with the postreceptor signaling pathways (sunitinib) provide us with the rational treatment options. These agents are generally used in combination with the standard chemotherapeutic drugs. Here, we discuss the basis of and the logic behind the use of these agents in the treatment of epithelial ovarian cancer, as well as their evaluation in different preclinical and clinical studies.

Significance of vascular endothelial growth factor in growth and peritoneal dissemination of ovarian cancer.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis which drives endothelial cell survival, proliferation, and migration while increasing vascular permeability. Playing an important role in the physiology of normal ovaries, VEGF has also been implicated in the pathogenesis of ovarian cancer. Essentially by promoting tumor angiogenesis and enhancing vascular permeability, VEGF contributes to the development of peritoneal carcinomatosis associated with malignant ascites formation, the characteristic feature of advanced ovarian cancer at diagnosis. In both experimental and clinical studies, VEGF levels have been inversely correlated with survival. Moreover, VEGF inhibition has been shown to inhibit tumor growth and ascites production and to suppress tumor invasion and metastasis. These findings have laid the basis for the clinical evaluation of agents targeting VEGF signaling pathway in patients with ovarian cancer. In this review, we will focus on VEGF involvement in the pathophysiology of ovarian cancer and its contribution to the disease progression and dissemination.