Doublecortin-like kinase 1 is a therapeutic target in squamous cell carcinoma.

Doublecortin like kinase 1 (DCLK1) plays a crucial role in several cancers including colon and pancreatic adenocarcinomas. However, its role in squamous cell carcinoma (SCC) remains unknown. To this end, we examined DCLK1 expression in head and neck SCC (HNSCC) and anal SCC (ASCC). We found that DCLK1 is elevated in patient SCC tissue, which correlated with cancer progression and poorer overall survival. Furthermore, DCLK1 expression is significantly elevated in human papilloma virus negative HNSCC, which are typically aggressive with poor responses to therapy. To understand the role of DCLK1 in tumorigenesis, we used specific shRNA to suppress DCLK1 expression. This significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on nonspecific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD (3,5-bis [2,4-difluorobenzylidene]-4-piperidone) binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth.

Anticancer Activity of Novel Difluorinated Curcumin Analog and Its Inclusion Complex with 2-Hydroxypropyl-ß-Cyclodextrin against Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the primary reason for cancer-related deaths in the US. Genetic mutations, drug resistance, the involvement of multiple signaling pathways, cancer stem cells (CSCs), and desmoplastic stroma, which hinders drug penetrance, contribute to poor chemotherapeutic efficacy. Hence, there is a need to identify novel drugs with improved delivery to improve treatment outcomes. Curcumin is one such compound that can inhibit multiple signaling pathways and CSCs. However, curcumin's clinical applicability for treating PDAC is limited because of its poor solubility in water and metabolic instability. Hence, we developed a difluorinated curcumin (CDF) analog that accumulates selectively in the pancreas and inhibits PDAC growth in vitro and in vivo. In the present work, we developed its 2-hydroxy-propyl-ß-cyclodextrin (HCD) inclusion complex to increase its water solubility and hydrolytic stability. The CDFHCD inclusion complex was characterized by spectroscopic, thermal, and microscopic techniques. The inclusion complex exhibited increased aqueous solubility, hydrolytic stability, and antiproliferative activity compared to parent CDF. Moreover, CDF and CDFHCD inhibited colony and spheroid formation, and induced cell cycle and apoptosis in PDAC cell lines. Hence, CDFHCD self-assembly is an efficient approach to increase water solubility and anticancer therapeutic efficacy, which now warrants advancement towards a clinical proof of concept in PDAC patients.

Infection-induced signals generated at the plasma membrane epigenetically regulate Wnt signaling in vitro and in vivo.

Wnt signaling regulates immunomodulatory functions during infection and inflammation. Employing NCCIT and HCT116 cells, having high endogenous Wnt signaling, we observed elevated levels of low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and Frizzled class receptor 10 (FZD10) and increases in ß-catenin, doublecortin-like kinase 1 (DCLK1), CD44 molecule (CD44), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). siRNA-induced knockdown of these receptors antagonized TOPflash reporter activity and spheroid growth in vitro and elevated Wnt-inhibitory factor 1 (WIF1) activity. Elevated mRNA and protein levels of LRP5/6 and FZD10 paralleled expression of WNT2b and WNT4 in colonic crypts at days 6 and 12 post-infection with Citrobacter rodentium (CR) and tended to decline at days 20-34. The CR mutant escV or the tankyrase inhibitor XAV939 attenuated these responses. A three-dimensional organoid assay in colonic crypts isolated from CR-infected mice revealed elevated levels of LRP5/6 and FZD10 and ß-catenin co-localization with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). Co-immunoprecipitation in the membrane fraction revealed that axin associates with LRP5/6 in CR-infected crypts, and this association was correlated with increased ß-catenin. Colon tumors from either CR-infected ApcPMin/+ or azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice had high LRP5/6 or FZD10 levels, and chronic Notch blockade through the γ-secretase inhibitor dibenzazepine down-regulated LRP5/6 and FZD10 expression. In CR-responsive CT-26 cells, siRNA-induced LRP5/6 or FZD10 knockdown antagonized TOPflash reporter activity. Elevated miR-153-3p levels correlated with LRP5/6 and FZD10, and miR-153-3p sequestration via a plasmid-based miR inhibitor system attenuated Wnt signaling. We conclude that infection-induced signals from the plasma membrane epigenetically regulate Wnt signaling.

Diphenylbutylpiperidine Antipsychotic Drugs Inhibit Prolactin Receptor Signaling to Reduce Growth of Pancreatic Ductal Adenocarcinoma in Mice.

BACKGROUND & AIMS: Prolactin (PRL) signaling is up-regulated in hormone-responsive cancers. The PRL receptor (PRLR) is a class I cytokine receptor that signals via the Janus kinase (JAK)-signal transducer and activator of transcription and mitogen-activated protein kinase pathways to regulate cell proliferation, migration, stem cell features, and apoptosis. Patients with pancreatic ductal adenocarcinoma (PDAC) have high plasma levels of PRL. We investigated whether PRLR signaling contributes to the growth of pancreatic tumors in mice. METHODS: We used immunohistochemical analyses to compare levels of PRL and PRLR in multitumor tissue microarrays. We used structure-based virtual screening and fragment-based drug discovery to identify compounds likely to bind PRLR and interfere with its signaling. Human pancreatic cell lines (AsPC-1, BxPC-3, Panc-1, and MiaPaCa-2), with or without knockdown of PRLR (clustered regularly interspaced short palindromic repeats or small hairpin RNA), were incubated with PRL or penfluridol and analyzed in proliferation and spheroid formation. C57BL/6 mice were given injections of UNKC-6141 cells, with or without knockdown of PRLR, into pancreas, and tumor development was monitored for 4 weeks, with some mice receiving penfluridol treatment for 21 days. Human pancreatic tumor tissues were implanted into interscapular fat pads of NSG mice, and mice were given injections of penfluridol daily for 28 days. Nude mice were given injections of Panc-1 cells, xenograft tumors were grown for 2 weeks, and mice were then given intraperitoneal penfluridol for 35 days. Tumors were collected from mice and analyzed by histology, immunohistochemistry, and immunoblots. RESULTS: Levels of PRLR were increased in PDAC compared with nontumor pancreatic tissues. Incubation of pancreatic cell lines with PRL activated signaling via JAK2-signal transducer and activator of transcription 3 and extracellular signal-regulated kinase, as well as formation of pancospheres and cell migration; these activities were not observed in cells with PRLR knockdown. Pancreatic cancer cells with PRLR knockdown formed significantly smaller tumors in mice. We identified several diphenylbutylpiperidine-class antipsychotic drugs as agents that decreased PRL-induced JAK2 signaling; incubation of pancreatic cancer cells with these compounds reduced their proliferation and formation of panco spheres. Injections of 1 of these compounds, penfluridol, slowed the growth of xenograft tumors in the different mouse models, reducing proliferation and inducing autophagy of the tumor cells. CONCLUSIONS: Levels of PRLR are increased in PDAC, and exposure to PRL increases proliferation and migration of pancreatic cancer cells. Antipsychotic drugs, such as penfluridol, block PRL signaling in pancreatic cancer cells to reduce their proliferation, induce autophagy, and slow the growth of xenograft tumors in mice. These drugs might be tested in patients with PDAC.

Targeting Major Signaling Pathways of Bladder Cancer with Phytochemicals: A Review.

Bladder cancer is the 9th most prevalent cancer worldwide and carries a protracted treatment course with significant patient expense, morbidity, and mortality. Over 95% of bladder cancers arise from the urothelium and invade into the underlying muscle layer before metastasizing. Trans-urethral resection and BCG therapy is the current first-line treatment for non-muscle invasive bladder cancer but carries a high rate of tumor recurrence and progression. The poor outcomes associated with advanced disease indicate the urgent need for new and improved treatment strategies. There is increasing investigation into the molecular signaling pathways involved in bladder cancer pathogenesis with the goal of uncovering potential therapeutic targets. This article reviews the major signaling pathways implicated in bladder cancer, including PI3K/AKT/mTOR, Ras/Raf/MEK/MAPK, NF-κB, Wnt/ß-catenin, Notch, Hedgehog, Hippo, JAK/STAT, and TGF-ß as well as major cellular receptors central to cancer pathophysiology, including EGFR, Her2, FGFR, and VEGF. We also discuss various naturally occurring phytochemicals that show evidence of targeting these molecular pathways including curcumin, resveratrol, green tea polyphenols, sulforaphane, erucin, genistein, genipin, baicalein, quercetin, isoquercitin, vitamin E, parthenolide, dioscin, triptolide, kaempferol, pterostilbene, isoliquiritigenin, and escin. This review highlights the potential use of these compounds in treatment of bladder cancer.

Dietary Interventions Ameliorate Infectious Colitis by Restoring the Microbiome and Promoting Stem Cell Proliferation in Mice.

Decreases in short-chain-fatty-acids (SCFAs) are linked to inflammatory bowel disease (IBD). Yet, the mechanisms through which SCFAs promote wound healing, orchestrated by intestinal stem cells, are poorly understood. We discovered that, in mice with Citrobacter rodentium (CR)-induced infectious colitis, treatment with Pectin and Tributyrin diets reduced the severity of colitis by restoring Firmicutes and Bacteroidetes and by increasing mucus production. RNA-seq in young adult mouse colon (YAMC) cells identified higher expression of Lgr4, Lgr6, DCLK1, Muc2, and SIGGIR after Butyrate treatment. Lineage tracing in CR-infected Lgr5-EGFP-IRES-CreERT2/ROSA26-LacZ (Lgr5-R) mice also revealed an expansion of LacZ-labeled Lgr5(+) stem cells in the colons of both Pectin and Tributyrin-treated mice compared to control. Interestingly, gut microbiota was required for Pectin but not Tributyrin-induced Lgr5(+) stem cell expansion. YAMC cells treated with sodium butyrate exhibited increased Lgr5 promoter reporter activity due to direct Butyrate binding with Lgr5 at -4.0 Kcal/mol, leading to thermal stabilization. Upon ChIP-seq, H3K4me3 increased near Lgr5 transcription start site that contained the consensus binding motif for a transcriptional activator of Lgr5 (SPIB). Thus, a multitude of effects on gut microbiome, differential gene expression, and/or expansion of Lgr5(+) stem cells seem to underlie amelioration of colitis following dietary intervention.

The Histone Demethylase KDM3A, Increased in Human Pancreatic Tumors, Regulates Expression of DCLK1 and Promotes Tumorigenesis in Mice.

BACKGROUND & AIMS: The histone lysine demethylase 3A (KDM3A) demethylates H3K9me1 and H3K9Me2 to increase gene transcription and is upregulated in tumors, including pancreatic tumors. We investigated its activities in pancreatic cancer cell lines and its regulation of the gene encoding doublecortin calmodulin-like kinase 1 (DCLK1), a marker of cancer stem cells. METHODS: We knocked down KDM3A in MiaPaCa-2 and S2-007 pancreatic cancer cell lines and overexpressed KDM3A in HPNE cells (human noncancerous pancreatic ductal cell line); we evaluated cell migration, invasion, and spheroid formation under hypoxic and normoxic conditions. Nude mice were given orthotopic injections of S2-007 cells, with or without (control) knockdown of KDM3A, and HPNE cells, with or without (control) overexpression of KDM3A; tumor growth was assessed. We analyzed pancreatic tumor tissues from mice and pancreatic cancer cell lines by immunohistochemistry and immunoblotting. We performed RNA-sequencing analysis of MiaPaCa-2 and S2-007 cells with knockdown of KDM3A and evaluated localization of DCLK1 and KDM3A by immunofluorescence. We analyzed the cancer genome atlas for levels of KDM3A and DCLK1 messenger RNA in human pancreatic ductal adenocarcinoma (PDAC) tissues and association with patient survival time. RESULTS: Levels of KDM3A were increased in human pancreatic tumor tissues and cell lines, compared with adjacent nontumor pancreatic tissues, such as islet and acinar cells. Knockdown of KDM3A in S2-007 cells significantly reduced colony formation, invasion, migration, and spheroid formation, compared with control cells, and slowed growth of orthotopic tumors in mice. We identified KDM3A-binding sites in the DCLK1 promoter; S2-007 cells with knockdown of KDM3A had reduced levels of DCLK1. HPNE cells that overexpressed KDM3A formed foci and spheres in culture and formed tumors and metastases in mice, whereas control HPNE cells did not. Hypoxia induced sphere formation and increased levels of KDM3A in S2-007 cells and in HPNE cells that overexpressed DCLK1, but not control HPNE cells. Levels of KDM3A and DCLK1 messenger RNA were higher in human PDAC than nontumor pancreatic tissues and correlated with shorter survival times of patients. CONCLUSIONS: We found human PDAC samples and pancreatic cancer cell lines to overexpress KDM3A. KDM3A increases expression of DCLK1, and levels of both proteins are increased in human PDAC samples. Knockdown of KDM3A in pancreatic cancer cell lines reduced their invasive and sphere-forming activities in culture and formation of orthotopic tumors in mice. Hypoxia increased expression of KDM3A in pancreatic cancer cells. Strategies to disrupt this pathway might be developed for treatment of pancreatic cancer.

Glycemic impact of a diet and lifestyle intervention on diabetics and prediabetics during treatment for non-muscle invasive bladder cancer.

Introduction: Patients with Type II Diabetes Mellitus (DM2) have increased risk of recurrence and progression of non-muscle invasive bladder cancer (NMIBC). Glucose control through lifestyle intervention is an uninvestigated, attractive strategy to decrease risk of cancer recurrence. We test the feasibility of a diet and exercise program and its glycemic impact in patients with DM2 and NMIBC.Materials/methods: Five participants with NMIBC and pre-diabetes or DM2 were recruited for a pilot, prospective clinical trial. Each participant received dietary counseling for 16 sessions during clinical visits. The intervention included a carbohydrate-restricted (CR) diet (<130 grams per day), 30 min, walking 5×/wk, and 5000 steps daily. Diet compliance was measured with 24-hour diet recall. Exercise was monitored with accelerometer and self-report.Results: Five participants enrolled and two participants completed the 12-month intervention. Adherence was 60% to CR diet and 84% to exercise goals. Participants reduced carbohydrate consumption by 44%. Participants showed reductions in fasting blood glucose, HbA1c, glucosuria, fasting blood insulin, and body weight, and increased euglycemia on continuous glucose monitoring.Conclusions: Adherence to a CR diet and exercise goals is feasible in patients with NMIBC and DM2 and also leads improved glucose control. A phase-II trial on bladder cancer-specific outcomes is warranted.

Preclinical Pharmacokinetics of Fosciclopirox, a Novel Treatment of Urothelial Cancers, in Rats and Dogs.

Pharmacokinetic studies in rats and dogs were performed to characterize the in vivo performance of a novel prodrug, fosciclopirox. Ciclopirox olamine (CPX-O) is a marketed topical antifungal agent with demonstrated in vitro and in vivo preclinical anticancer activity in several solid tumor and hematologic malignancies. The oral route of administration for CPX-O is not feasible due to low bioavailability and dose-limiting gastrointestinal toxicities. To enable parenteral administration, the phosphoryl-oxymethyl ester of ciclopirox (CPX), fosciclopirox (CPX-POM), was synthesized and formulated as an injectable drug product. In rats and dogs, intravenous CPX-POM is rapidly and completely metabolized to its active metabolite, CPX. The bioavailability of the active metabolite is complete following CPX-POM administration. CPX and its inactive metabolite, ciclopirox glucuronide (CPX-G), are excreted in urine, resulting in delivery of drug to the entire urinary tract. The absolute bioavailability of CPX following subcutaneous administration of CPX-POM is excellent in rats and dogs, demonstrating the feasibility of this route of administration. These studies confirmed the oral bioavailability of CPX-O is quite low in rats and dogs compared with intravenous CPX-POM. Given its broad-spectrum anticancer activity in several solid tumor and hematologic cancers and renal elimination, CPX-POM is being developed for the treatment of urothelial cancer. The safety, dose tolerance, pharmacokinetics, and pharmacodynamics of intravenous CPX-POM are currently being characterized in a United States multicenter first-in-human Phase 1 clinical trial in patients with advanced solid tumors (NCT03348514).

Pleotropic role of RNA binding protein CELF2 in autophagy induction.

We previously reported that ionizing radiation (IR) mediates cell death through the induction of CUGBP elav-like family member 2 (CELF2), a tumor suppressor. CELF2 is an RNA binding protein that modulates mRNA stability and translation. Since IR induces autophagy, we hypothesized that CELF2 regulates autophagy-mediated colorectal cancer (CRC) cell death. For clinical relevance, we determined CELF2 levels in The Cancer Genome Atlas (TCGA). Role of CELF2 in radiation response was carried out in CRC cell lines by immunoblotting, immunofluorescence, autophagic vacuole analyses, RNA stability assay, quantitative polymerase chain reaction and electron microscopy. In vivo studies were performed in a xenograft tumor model. TCGA analyses demonstrated that compared to normal tissue, CELF2 is expressed at significantly lower levels in CRC, and is associated with better overall 5-year survival in patients receiving radiation. Mechanistically, CELF2 increased levels of critical components of the autophagy cascade including Beclin-1, ATG5, and ATG12 by modulating mRNA stability. CELF2 also increased autophagic flux in CRC. IR significantly induced autophagy in CRC which correlates with increased levels of CELF2 and autophagy associated proteins. Silencing CELF2 with siRNA, mitigated IR induced autophagy. Moreover, knockdown of CELF2 in vivo conferred tumor resistance to IR. These studies elucidate an unrecognized role for CELF2 in inducing autophagy and potentiating the effects of radiotherapy in CRC.

Targeting cancer stem cells and signaling pathways by phytochemicals: Novel approach for breast cancer therapy.

Breast cancer is the most common form of cancer diagnosed in women worldwide and the second leading cause of cancer-related deaths in the USA. Despite the development of newer diagnostic methods, selective as well as targeted chemotherapies and their combinations, surgery, hormonal therapy, radiotherapy, breast cancer recurrence, metastasis and drug resistance are still the major problems for breast cancer. Emerging evidence suggest the existence of cancer stem cells (CSCs), a population of cells with the capacity to self-renew, differentiate and be capable of initiating and sustaining tumor growth. In addition, CSCs are believed to be responsible for cancer recurrence, anticancer drug resistance, and metastasis. Hence, compounds targeting breast CSCs may be better therapeutic agents for treating breast cancer and control recurrence and metastasis. Naturally occurring compounds, mainly phytochemicals have gained immense attention in recent times because of their wide safety profile, ability to target heterogeneous populations of cancer cells as well as CSCs, and their key signaling pathways. Therefore, in the present review article, we summarize our current understanding of breast CSCs and their signaling pathways, and the phytochemicals that affect these cells including curcumin, resveratrol, tea polyphenols (epigallocatechin-3-gallate, epigallocatechin), sulforaphane, genistein, indole-3-carbinol, 3, 3'-di-indolylmethane, vitamin E, retinoic acid, quercetin, parthenolide, triptolide, 6-shogaol, pterostilbene, isoliquiritigenin, celastrol, and koenimbin. These phytochemicals may serve as novel therapeutic agents for breast cancer treatment and future leads for drug development.

Stromal contributions to the carcinogenic process.

Tumor-associated stromal cells are dynamic characters that endorse the carcinogenic process in a multitude of ways. The tumor microenvironment plays a crucial role throughout the tumor progression, which includes initiation, growth, invasion, and metastasis. The tumor microenvironment consists of cellular and non-cellular components. Tumor-associated stromal cell types include the microbiome, immune cells including macrophages, dendritic and T-cells, cells associated with blood and lymphatic vessels including pericytes and endothelial cells, fibroblasts, neuronal cells, and adipocytes. The non-cellular components of the microenvironment include matrix proteins and secreted factors. The development of therapies that target the mechanisms by which stromal cells contribute to successful tumorigenesis is major goal of upcoming cancer research. The purpose of this review is to present a comprehensive discussion of the role of each of the tumor-associated stromal cell types in the carcinogenic process with a special focus on target development and therapeutic intervention. © 2016 Wiley Periodicals, Inc.

An ornamental plant targets epigenetic signaling to block cancer stem cell-driven colon carcinogenesis.

Phytochemicals modulate key cellular signaling pathways and have proven anticancer effects. Alcea rosea(AR; Hollyhock) is an ornamental plant with known anti-inflammatory properties. This study explored its role as an anticancer agent. The AR seed extract (AR extract) inhibited proliferation and colony formation in a dose- and time-dependent manner and promoted apoptosis as was evidenced by cleavage of PARP and increased expression of Bax accompanying reduced levels of BCL-xl protein in HCT116 and SW480 cells, respectively. In addition, AR extract-arrested cells at Go/G1 phase of cell cycle and exhibited decreases in Cyclin D1. AR extract-treated cells exhibited reduced number and size of colonospheres in a dose-dependent manner concomitant with decreases in cancer stem cell (CSC) markers ALDH1A1 and Dclk1. Relative levels of ß-catenin, Notch-ICD, Hes1 and EZH2 were also attenuated by AR extract. TOP-flash reporter activity, a measure of Wnt signaling, decreased significantly in response to treatment while overexpression of wild type but not mutant EZH2, reversed the inhibitory effects. Moreover, WIF1 (a Wnt antagonist) promoter activity increased dramatically following treatment with AR extract which phenocopied increases in WIF1 reporter activity following EZH2 knockdown.In vivo, AR extract attenuated tumor growth due probably to reduced levels of EZH2, ß-catenin, CyclinD1 and Ki-67 along with reduced levels of CSC markers. Since partial purification via HPLC yielded a prominent peak, efforts are underway to identify the active ingredient(s). Taken together, the results clearly suggest that AR extract/active component(s) can be an effective preventative/therapeutic agent to target colon cancer.

RNA binding protein RBM3 increases ß-catenin signaling to increase stem cell characteristics in colorectal cancer cells.

Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. It arises from loss of intestinal epithelial homeostasis and hyperproliferation of the crypt epithelium. In order to further understand the pathogenesis of CRC it is important to further understand the factors regulating intestinal epithelial proliferation and more specifically, regulation of the intestinal epithelial stem cell compartment. Here, we investigated the role of the RNA binding protein RBM3 in stem cell homeostasis in colorectal cancers. Using a doxycycline (Dox) inducible RBM3 overexpressing cell lines HCT 116 and DLD-1, we measured changes in side population (SP) cells that have high xenobiotic efflux capacity and increased capacity for self-renewal. In both cell lines, RBM3 induction showed significant increases in the percentage of side population cells. Additionally, we observed increases in spheroid formation and in cells expressing DCLK1, LGR5 and CD44Hi . As the Wnt/ß-catenin signaling pathway is important for both physiologic and cancer stem cells, we next investigated the effects of RBM3 overexpression on ß-catenin activity. RBM3 overexpression increased levels of nuclear ß-catenin as well as TCF/LEF transcriptional activity. In addition, there was inactivation of GSK3ß leading to decreased ß-catenin phosphorylation. Pharmacologic inhibition of GSK3ß using (2'Z,3'E)-6-Bromoindirubin-3'-oxime (BIO) also recapitulates the RBM3 induced ß-catenin activity. In conclusion, we see that RNA binding protein RBM3 induces stemness in colorectal cancer cells through a mechanism involving suppression of GSK3ß activity thereby enhancing ß-catenin signaling. © 2015 Wiley Periodicals, Inc.

Honokiol inhibits melanoma stem cells by targeting notch signaling.

Melanoma is an aggressive disease with limited therapeutic options. Here, we determined the effects of honokiol (HNK), a biphenolic natural compound on melanoma cells and stemness. HNK significantly inhibited melanoma cell proliferation, viability, clonogenicity and induced autophagy. In addition, HNK significantly inhibited melanosphere formation in a dose dependent manner. Western blot analyses also demonstrated reduction in stem cell markers CD271, CD166, Jarid1b, and ABCB5. We next examined the effect of HNK on Notch signaling, a pathway involved in stem cell self-renewal. Four different Notch receptors exist in cells, which when cleaved by a series of enzymatic reactions catalyzed by Tumor Necrosis Factor-α-Converting Enzyme (TACE) and γ-secretase protein complex, results in the release of the Notch intracellular domain (NICD), which then translocates to the nucleus and induces target gene expression. Western blot analyses demonstrated that in HNK treated cells there is a significant reduction in the expression of cleaved Notch-2. In addition, there was a reduction in the expression of downstream target proteins, Hes-1 and cyclin D1. Moreover, HNK treatment suppressed the expression of TACE and γ-secretase complex proteins in melanoma cells. To confirm that suppression of Notch-2 activation is critical for HNK activity, we overexpressed NICD1, NICD2, and performed HNK treatment. NICD2, but not NICD1, partially restored the expression of Hes-1 and cyclin D1, and increased melanosphere formation. Taken together, these data suggest that HNK is a potent inhibitor of melanoma cells, in part, through the targeting of melanoma stem cells by suppressing Notch-2 signaling.

Prolactin signaling enhances colon cancer stemness by modulating Notch signaling in a Jak2-STAT3/ERK manner.

Prolactin (PRL) is a secretory cytokine produced by various tissues. Binding to the cognate PRL receptor (PRLR), it activates intracellular signaling via janus kinase (JAK), extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription (STAT) proteins. PRL regulates diverse activities under normal and abnormal conditions, including malignancies. Previous clinical data suggest serum PRL levels are elevated in colorectal cancer (CRC) patients. In this study, we first determined the expression of PRL and PRLR in colon cancer tissue and cell lines. Higher levels of PRLR expression were observed in the cancer cells and cell lines compared with normal colonic epithelial cells. Incubation of colon cancer cells with PRL-induced JAK2, STAT3 and ERK1/2 phosphorylation and increased expression of Jagged 1, which is a Notch-1 receptor ligand. Notch signaling regulates CRC stem cell population. We observed increased accumulation of the cleaved/active form of Notch-1 receptor (Notch intracellular domain) and increased expression of Notch responsive genes HEY1, HES1 and stem cell marker genes DCLK1, LGR5, ALDH1 and CD44. Finally, inhibiting PRL induced JAK2-STAT3 and JAK2-ERK1/2 using AG490 and PD98059, respectively, leads to complete abrogation of Notch signaling, suggesting a role for this pathway in regulating CRC stem cells. Together, our results demonstrate that cytokine signaling induced by PRL is active in colorectal cancers and may provide a novel target for therapeutic intervention.

Reg4-induced mitogenesis involves Akt-GSK3ß-ß-Catenin-TCF-4 signaling in human colorectal cancer.

Upregulation of regenerating gene 4 (Reg4) is observed in many human gastrointestinal malignancies including colorectal cancer (CRC). We previously reported a Reg4-mediated induction of epidermal growth factor receptor-Akt-AP1 signaling regulating CRC cell apoptosis. However, the role of Reg4 in the regulation of CRC cell division is poorly understood. This study tests the hypothesis that Reg4 induces Akt-GSK3ß-ß-Catenin-TCF-4 signaling to regulate CRC cell division. In vitro models of human CRC were used to determine the role of Reg4 in regulation of CRC cell division. Cell cycle studies demonstrated that Reg4 treatment significantly decreased CRC cell number in G1 phase and increased in G2 phase. Subsequently Reg4 significantly increased the mitotic index of CRC cells. As assessed by real-time RT-PCR and Western blot analyses, Reg4 significantly increased the expression of cell cycle regulatory genes Cyclin D1 and D3, and associated Cyclin-dependent kinases (CDK4 and CDK6). Reg4-mediated increase in these genes involved a pathway that included an induced Akt activity by increasing phosphorylation of Thr308 and Ser473, a reduced glycogen synthase kinase 3ß (GSK-3ß) activity by increasing phosphorylation of Ser9, an induced nuclear translocation of ß-Catenin by decreasing phosphorylation of Ser33/37/Thr41, and an increased TCF-4 transcriptional activity. Furthermore, antagonism of Reg4-signaling using Reg4-specific mAbs (2H6 and 3E5) and Akt inhibitor significantly decreased, whereas agonism using GSK-3ß antagonist (SB216763) significantly increased mitotic index and proliferation of CRC cells. These results identify Reg4 as a key regulator of the CRC cell division and proliferation, hence a potential target of human CRC treatment.

Role of STAT3 in pancreatic cancer.

Pancreatic cancer remains a serious and deadly disease, impacting people globally. There remain prominent gaps in the current understanding of the disease, specifically regarding the role of the signal transducer and activator of transcription (STAT) family of proteins in pancreatic tumors. STAT proteins, particularly STAT3, play important roles in pancreatic cancer, especially pancreatic ductal adenocarcinoma (PDAC), which is the most prevalent histotype. The role of STAT3 across a continuum of molecular processes, such as PDAC tumorigenesis and progression, immune escape, drug resistance and stemness, and modulation of the tumor microenvironment (TME), are only a tip of the iceberg. In some ways, the role of STAT3 in PDAC may hold greater importance than that of oncogenic Kirsten rat sarcoma virus (KRAS). This makes STAT3 a highly attractive target for developing targeted therapies for the treatment of pancreatic cancer. In this review, the current knowledge of STAT3 in pancreatic cancer has been summarized, particularly relating to STAT3 activation in cancer cells, cells of the TME, and the state of targeting STAT3 in pre-clinical and clinical trials of PDAC.

DCLK1-Mediated Regulation of Invadopodia Dynamics and Matrix Metalloproteinase Trafficking Drives Invasive Progression in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a major health concern due to its high mortality from poor treatment responses and locoregional tumor invasion into life sustaining structures in the head and neck. A deeper comprehension of HNSCC invasion mechanisms holds the potential to inform targeted therapies that may enhance patient survival. We previously reported that doublecortin like kinase 1 (DCLK1) regulates invasion of HNSCC cells. Here, we tested the hypothesis that DCLK1 regulates proteins within invadopodia to facilitate HNSCC invasion. Invadopodia are specialized subcellular protrusions secreting matrix metalloproteinases that degrade the extracellular matrix (ECM). Through a comprehensive proteome analysis comparing DCLK1 control and shDCLK1 conditions, our findings reveal that DCLK1 plays a pivotal role in regulating proteins that orchestrate cytoskeletal and ECM remodeling, contributing to cell invasion. Further, we demonstrate in TCGA datasets that DCLK1 levels correlate with increasing histological grade and lymph node metastasis. We identified higher expression of DCLK1 in the leading edge of HNSCC tissue. Knockdown of DCLK1 in HNSCC reduced the number of invadopodia, cell adhesion and colony formation. Using super resolution microscopy, we demonstrate localization of DCLK1 in invadopodia and colocalization with mature invadopodia markers TKS4, TKS5, cortactin and MT1-MMP. We carried out phosphoproteomics and validated using immunofluorescence and proximity ligation assays, the interaction between DCLK1 and motor protein KIF16B. Pharmacological inhibition or knockdown of DCLK1 reduced interaction with KIF16B, secretion of MMPs, and cell invasion. This research unveils a novel function of DCLK1 within invadopodia to regulate the trafficking of matrix degrading cargo. The work highlights the impact of targeting DCLK1 to inhibit locoregional invasion, a life-threatening attribute of HNSCC.

Selective targeting of IRAK1 attenuates low molecular weight hyaluronic acid-induced stemness and non-canonical STAT3 activation in epithelial ovarian cancer.

Advanced epithelial ovarian cancer (EOC) survival rates are dishearteningly low, with ~25% surviving beyond 5 years. Evidence suggests that cancer stem cells contribute to acquired chemoresistance and tumor recurrence. Here, we show that IRAK1 is upregulated in EOC tissues, and enhanced expression correlates with poorer overall survival. Moreover, low molecular weight hyaluronic acid, which is abundant in malignant ascites from patients with advanced EOC, induced IRAK1 phosphorylation leading to STAT3 activation and enhanced spheroid formation. Knockdown of IRAK1 impaired tumor growth in peritoneal disease models, and impaired HA-induced spheroid growth and STAT3 phosphorylation. Finally, we determined that TCS2210, a known inducer of neuronal differentiation in mesenchymal stem cells, is a selective inhibitor of IRAK1. TCS2210 significantly inhibited EOC growth in vitro and in vivo both as monotherapy, and in combination with cisplatin. Collectively, these data demonstrate IRAK1 as a druggable target for EOC.