High Oestrogen receptor alpha expression correlates with adverse prognosis and promotes metastasis in colorectal cancer.

In normal colon tissue, oestrogen receptor alpha (ERα) is expressed at low levels, while oestrogen receptor beta (ERß) is considered the dominant subtype. However, in colon carcinomas, the ERα/ß ratio is often increased, an observation that prompted us to further investigate ERα's role in colorectal cancer (CRC). Here, we assessed ERα nuclear expression in 351 CRC patients. Among them, 119 exhibited positive ERα nuclear expression, which was significantly higher in cancer tissues than in matched normal tissues. Importantly, patients with positive nuclear ERα expression had a poor prognosis. Furthermore, positive ERα expression correlated with increased levels of the G-protein coupled cysteinyl leukotriene receptor 1 (CysLT1R) and nuclear ß-catenin, both known tumour promoters. In mouse models, ERα expression was decreased in Cysltr1-/- CAC (colitis-associated colon cancer) mice but increased in ApcMin/+ mice with wild-type Cysltr1. In cell experiments, an ERα-specific agonist (PPT) increased cell survival via WNT/ß-catenin signalling. ERα activation also promoted metastasis in a zebrafish xenograft model by affecting the tight junction proteins ZO-1 and Occludin. Pharmacological blockade or siRNA silencing of ERα limited cell survival and metastasis while restoring tight junction protein expression. In conclusion, these findings highlight the potential of ERα as a prognostic marker for CRC and its role in metastasis.

The tumor promoter cysteinyl leukotriene receptor 1 regulates PD-L1 expression in colon cancer cells via the Wnt/ß-catenin signaling axis.

Immunotherapy targeting programmed death-ligand 1 (PD-L1) or PD-1 in solid tumors has been shown to be clinically beneficial. However, in colorectal cancer (CRC), only a subset of patients benefit from PD-1/PD-L1 treatment. Previously, we showed that high cysteinyl leukotriene receptor 1 (CysLT1R) levels are associated with poor prognosis in CRC patients. Recently, we have revealed the role of the tumor promoter CysLT1R in drug resistance and stemness in colon cancer (CC) cells. Here, we show the role of the CysLT1R/Wnt/ß-catenin signaling axis in the regulation of PD-L1 using both in vitro and in vivo preclinical model systems. Interestingly, we found that both endogenous and IFNγ-induced PD-L1 expression in CC cells is mediated through upregulation of CysLT1R, which enhances Wnt/ß-catenin signaling. Therapeutic targeting of CysLT1R with its antagonist montelukast (Mo), as well as CRISPR/Cas9-mediated or doxycycline-inducible functional absence of CysLT1R, negatively regulated PD-L1 expression in CC cells. Interestingly, an anti-PD-L1 neutralizing antibody exhibited stronger effects together with the CysLT1R antagonist in cells (Apcmut or CTNNB1mut) with either endogenous or IFNγ-induced PD-L1 expression. Additionally, mice treated with Mo showed depletion of PD-L1 mRNA and protein. Moreover, in CC cells with combined treatment of a Wnt inhibitor and an anti-PD-L1 antibody was effective only in ß-catenin-dependent (APCmut) context. Finally, analysis of public dataset showed positive correlations between the PD-L1 and CysLT1R mRNA levels. These results elucidate a previously underappreciated CysLT1R/Wnt/ß-catenin signaling pathway in the context of PD-L1 inhibition in CC, which might be considered for improving the efficacy of anti-PD-L1 therapy in CC patients. Video Abstract.

DNA Methylation and Gene Expression of the Cysteinyl Leukotriene Receptors as a Prognostic and Metastatic Factor for Colorectal Cancer Patients.

Colorectal cancer (CRC), one of the leading causes of cancer-related deaths in the western world, is the third most common cancer for both men and women. As a heterogeneous disease, colon cancer (CC) is caused by both genetic and epigenetic changes. The prognosis for CRC is affected by a variety of features, including late diagnosis, lymph node and distant metastasis. The cysteinyl leukotrienes (CysLT), as leukotriene D4 and C4 (LTD4 and LTC4), are synthesized from arachidonic acid via the 5-lipoxygenase pathway, and play an important role in several types of diseases such as inflammation and cancer. Their effects are mediated via the two main G-protein-coupled receptors, CysLT1R and CysLT2R. Multiple studies from our group observed a significant increase in CysLT1R expression in the poor prognosis group, whereas CysLT2R expression was higher in the good prognosis group of CRC patients. Here, we systematically explored and established the role of the CysLTRs, cysteinyl leukotriene receptor 1(CYSLTR1) and cysteinyl leukotriene receptor 2 (CYSLTR2) gene expression and methylation in the progression and metastasis of CRC using three unique in silico cohorts and one clinical CRC cohort. Primary tumor tissues showed significant CYSLTR1 upregulation compared with matched normal tissues, whereas it was the opposite for the CYSLTR2. Univariate Cox proportional-hazards (CoxPH) analysis yielded a high expression of CYSLTR1 and accurately predicted high-risk patients in terms of overall survival (OS; hazard ratio (HR) = 1.87, p = 0.03) and disease-free survival [DFS] Hazard ratio [HR] = 1.54, p = 0.05). Hypomethylation of the CYSLTR1 gene and hypermethylation of the CYSLTR2 gene were found in CRC patients. The M values of the CpG probes for CYSLTR1 are significantly lower in primary tumor and metastasis samples than in matched normal samples, but those for CYSLTR2 are significantly higher. The differentially upregulated genes between tumor and metastatic samples were uniformly expressed in the high-CYSLTR1 group. Two epithelial-mesenchymal transition (EMT) markers, E-cadherin (CDH1) and vimentin (VIM) were significantly downregulated and upregulated in the high-CYSLTR1 group, respectively, but the result was opposite to that of CYSLTR2 expression in CRC. CDH1 expression was high in patients with less methylated CYSLTR1 but low in those with more methylated CYSLTR2. The EMT-associated observations were also validated in CC SW620 cell-derived colonospheres, which showed decreased E-cadherin expression in the LTD4 stimulated cells, but not in the CysLT1R knockdown SW620 cells. The methylation profiles of the CpG probes for CysLTRs significantly predicted lymph node (area under the curve [AUC] = 0.76, p < 0.0001) and distant (AUC = 0.83, p < 0.0001) metastasis. Intriguingly, the CpG probes cg26848126 (HR = 1.51, p = 0.03) for CYSLTR1, and cg16299590 (HR = 2.14, p = 0.03) for CYSLTR2 significantly predicted poor prognosis in terms of OS, whereas the CpG probe cg16886259 for CYSLTR2 significantly predicts a poor prognosis group in terms of DFS (HR = 2.88, p = 0.03). The CYSLTR1 and CYSLTR2 gene expression and methylation results were successfully validated in a CC patient cohort. In this study, we have demonstrated that CysLTRs' methylation and gene expression profile are associated with the progression, prognosis, and metastasis of CRC, which might be used for the assessment of high-risk CRC patients after validating the result in a larger CRC cohort.

Increased MARCKS Activity in BRAF Inhibitor-Resistant Melanoma Cells Is Essential for Their Enhanced Metastatic Behavior Independent of Elevated WNT5A and IL-6 Signaling.

Treatment of melanoma with a BRAF inhibitor (BRAFi) frequently initiates development of BRAFi resistance, leading to increased tumor progression and metastasis. Previously, we showed that combined inhibition of elevated WNT5A and IL-6 signaling reduced the invasion and migration of BRAFi-resistant (BRAFi-R) melanoma cells. However, the use of a combined approach per se and the need for high inhibitor concentrations to achieve this effect indicate a need for an alternative and single target. One such target could be myristoylated alanine-rich C-kinase substrate (MARCKS), a downstream target of WNT5A in BRAFi-sensitive melanoma cells. Our results revealed that MARCKS protein expression and activity are significantly elevated in PLX4032 and PLX4720 BRAFi-R A375 and HTB63 melanoma cells. Surprisingly, neither WNT5A nor IL-6 contributed to the increases in MARCKS expression and activity in BRAFi-R melanoma cells, unlike in BRAFi-sensitive melanoma cells. However, despite the above findings, our functional validation experiments revealed that MARCKS is essential for the increased metastatic behavior of BRAFi-R melanoma cells. Knockdown of MARCKS in BRAFi-R melanoma cells caused reductions in the F-actin content and the number of filopodia-like protrusions, explaining the impaired migration, invasion and metastasis of these cells observed in vitro and in an in vivo zebrafish model. In our search for an alternative explanation for the increased activity of MARCKS in BRAFi-R melanoma cells, we found elevated basal activities of PKCα, PKCε, PKCι, and RhoA. Interestingly, combined inhibition of basal PKC and RhoA effectively impaired MARCKS activity in BRAFi-R melanoma cells. Our results reveal that MARCKS is an attractive single antimetastatic target in BRAFi-R melanoma cells.

Combined Estrogen Alpha and Beta Receptor Expression Has a Prognostic Significance for Colorectal Cancer Patients.

We reported that high estrogen receptor beta (ERß) expression is independently associated with better prognosis in female colorectal cancer (CRC) patients. However, estrogen receptor alpha (ERα) is expressed at very low levels in normal colon mucosa, and its prognostic role in CRC has not been explored. Herein, we investigated the combined role of ERα and ERß expression in the prognosis of female patients with CRC, which, to the best of our knowledge, is the first study to investigate this topic. A total number of 306 primary CRCs were immunostained for ERα and ERß expression. A Cox regression model was used to evaluate overall survival (OS) and disease-free survival (DFS). The combined expression of high ERß + negative ERα correlates with longer OS (HR = 0.23; 95% CI: 0.11-0.45, P <0.0001) and DFS (HR = 0.10; 95% CI: 0.03-0.26, P < 0.0001) and a more favorable tumor outcome, as well as significantly higher expression of antitumorigenic proteins than combined expression of low ERß + positive ERα. Importantly, we found that low ERß expression was associated with local recurrence of CRC, whereas ERα expression was correlated with liver metastasis. Overall, our results show that the combined high ERß + negative ERα expression correlated with a better prognosis for CRC patients. Our results suggest that the combined expression of ERα and ERß could be used as a predictive combination marker for CRC patients, especially for predicting DFS.

Identification of a Novel Five-Gene Signature as a Prognostic and Diagnostic Biomarker in Colorectal Cancers.

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide. The current TNM (Tumor, Node, and Metastasis) classification approach is suboptimal in determining the prognosis of CRC patients. The prognosis for CRC is affected by a variety of features that are present at the initial diagnosis. Herein, we performed a systematic exploration and established a novel five-panel gene signature as a prognostic and early diagnosis biomarker after performing differential gene expression analyses in five independent in silico CRCs cohort and independently validating it in one clinical cohort, using immunohistochemistry. Four genes (BDNF, PTGS2, GSK3B, and CTNNB1) were significantly upregulated and one gene (HPGD) was significantly downregulated in primary tumor tissues compared with adjacent normal tissues throughout all the five in silico datasets. The univariate CoxPH analysis yielded a five-gene signature that accurately predicted overall survival (OS) and recurrence-free survival (RFS) in the in silico training (AUC = 0.73 and 0.69, respectively) and one independent in silico validation cohort (AUC = 0.69 and 0.74, respectively). This five-gene signature demonstrated significant associations with poor OS in independent clinical validation cohorts of colon cancer (CC) patients (AUC = 0.82). Intriguingly, a risk stratification model comprising of the five-gene signature together with TNM stage and gender status achieved an even superior AUC of 0.89 in the clinical cohorts. On the other hand, the circulating mRNA expression of the upregulated four-gene signature achieved a robust AUC = 0.83 with high sensitivity and specificity as a diagnosis marker in plasma from CRC patients. We have identified a novel, five-gene signature as an independent predictor of OS, which in combination with TNM stage and gender offers an easy-to-translate and facile assay for the personalized risk-assessment in CRC patients.

High PGD2 receptor 2 levels are associated with poor prognosis in colorectal cancer patients and induce VEGF expression in colon cancer cells and migration in a zebrafish xenograft model.

BACKGROUND: Despite intense research, the prognosis for patients with advanced colorectal cancer (CRC) remains poor. The prostaglandin D2 receptors DP1 and DP2 are explored here as potential therapeutic targets for advanced CRC. METHODS: A CRC cohort was analysed to determine whether DP1 and DP2 receptor expression correlates with patient survival. Four colon cancer cell lines and a zebrafish metastasis model were used to explore how DP1/DP2 receptor expression correlates with CRC progression. RESULTS: Analysis of the clinical CRC cohort revealed high DP2 expression in tumour tissue, whereas DP1 expression was low. High DP2 expression negatively correlated with overall survival. Other pathological indicators, such as TNM stage and metastasis, positively correlated with DP2 but not DP1 expression. In accordance, the in vitro results showed high DP2 expression in four CC-cell lines, but only one expressed DP1. DP2 stimulation resulted in increased proliferation, p-ERK1/2 and VEGF expression/secretion. DP2-stimulated cells exhibited increased migration in the zebrafish metastasis model. CONCLUSION: Our results support DP2 receptor expression and signalling as a therapeutic target in CRC progression based on its expression in CRC tissue correlating with poor patient survival and that it triggers proliferation, p-ERK1/2 and VEGF expression and release and increased metastatic activity in CC-cells.

Brain-Derived Neurotrophic Factor, Neutrophils and Cysteinyl Leukotriene Receptor 1 as Potential Prognostic Biomarkers for Patients with Colon Cancer.

The tumor microenvironment has been recognized as a complex network in which immune cells play an important role in cancer progression. We found significantly higher CD66b neutrophil expression in tumor tissue than in matched normal mucosa in the Malmö colon cancer (CC) cohort and poorer survival of stage I-III patients with high CD66b expression. Additionally, mice lacking CysLT1R expression (cysltr1-/-) produce less brain-derived neurotrophic factor (BDNF) compared to WT mice and Montelukast (a CysLT1R antagonist)-treated mice also reduced BDNF expression in a mouse xenograft model with human SW480 CC cells. CD66b and BDNF expression was significantly higher in patient tumor tissues than in the matched normal mucosa. The univariate Cox PH analysis yielded CD66b and BDNF as an independent predictor of overall survival, which was also found in the public TCGA-COAD dataset. We also discovered a strong positive correlation between CD66b, BDNF and CysLT1R expression in the Malmö CC cohort and in the TCGA-COAD dataset. Our data suggest that CD66b/BDNF/CysLT1R expression as a prognostic combined biomarker signature for CC patients.

Tumour suppressor 15-hydroxyprostaglandin dehydrogenase induces differentiation in colon cancer via GLI1 inhibition.

Inflammation is an established risk factor for colorectal cancer. We and others have shown that colorectal cancer patients with elevated cysteinyl leukotriene receptor 2 (CysLT2R) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) levels exhibit good prognoses. However, both CysLT2R and 15-PGDH, which act as tumour suppressors, are often suppressed in colorectal cancer. We previously reported that leukotriene C4 (LTC4)-induced differentiation in colon cancer via CysLT2R signalling. Here, we investigated the involvement of Hedgehog (Hh)-GLI1 signalling, which is often hyperactivated in colorectal cancer. We found that the majority of colorectal cancer patients had high-GLI1 expression, which was negatively correlated with CysLT2R, 15-PGDH, and Mucin-2 and overall survival compared with the low-GLI1 group. LTC4-induced 15-PGDH downregulated both the mRNA and protein expression of GLI1 in a protein kinase A (PKA)-dependent manner. Interestingly, the LTC4-induced increase in differentiation markers and reduction in Wnt targets remained unaltered in GLI1-knockdown cells. The restoration of GLI1 in 15-PGDH-knockdown cells did not ameliorate the LTC4-induced effects, indicating the importance of both 15-PGDH and GLI1. LTC4-mediated reduction in the DCLK1 and LGR5 stemness markers in colonospheres was abolished in cells lacking 15-PGDH or GLI1. Both DCLK1 and LGR5 were highly increased in tumour tissue compared with the matched controls. Reduced Mucin-2 levels were observed both in zebrafish xenografts with GLI1-knockdown cells and in the cysltr2-/- colitis-associated colon cancer (CAC) mouse model. Furthermore, GLI1 expression was positively correlated with stemness and negatively correlated with differentiation in CRC patients when comparing tumour and mucosal tissues. In conclusion, restoring 15-PGDH expression via CysLT2R activation might benefit colorectal cancer patients.

Cysteinyl leukotriene receptor 1 promotes 5-fluorouracil resistance and resistance-derived stemness in colon cancer cells.

Colon cancer is a therapy-resistant cancer with a low 5-year survival frequency. The drug 5-fluorouracil (5-FU) has been used as a first-line therapy in metastatic colon cancer in combination with leucovorin or oxaliplatin with a >40% resistance rate. High CysLT1R expression in tumors is associated with poor survival of colon cancer patients. We sought to examine the role of CysLT1R in 5-FU resistance and established 5-FU-resistant (5-FU-R) colon cancer cells. These 5-FU-R-cells expressed increased levels of CysLT1R and showed increased survival and migration compared to nonresistant cells. Increases in thymidylate synthase and active ß-catenin were also observed in the 5-FU-R-cells. LTD4/CysLT1R signaling was further increased and abolished after CYSLTR1 CRISPR-Cas9-knockdown and reduced in CysLT1R-doxycycline-knockdown experiments and CysLT1R-antagonist montelukast/5-FU-treated cells. Montelukast and 5-FU resulted in synergistic effects by reducing HT-29 cell and 5-FU-R-HT-29 cell migration and zebrafish xenograft metastasis. An increase in the stem cell markers in 5-FU-R-cells and 5-FU-R-cell-derived colonospheres and in CysLT1R-Dox-knockdown cells increased colonosphere formation and stem cell markers was noticed after 5-FU treatment. IL-4-mediated stemness in both HT-29-colonospheres and 5-FU-R-cell derived colonospheres was abolished by montelukast or montelukast + 5-FU-treatment. Targeting CysLT1R signaling by montelukast might reverse drug resistance and decrease resistance-derived stemness in colon cancer patients.

Tumour-suppressive effect of oestrogen receptor ß in colorectal cancer patients, colon cancer cells, and a zebrafish model.

Oestrogen receptor ß (ERß) has been suggested to have anti-proliferative and anti-tumour effects in breast and prostate cancer cells, but other studies have indicated its tumour-promoting effects. Understanding the complex effects of this receptor in different contexts requires further study. We reported that high ERß expression is independently associated with improved prognosis in female colorectal cancer (CRC) patients. Herein, we investigated the possible anti-tumour effect of ERß and its selective agonist. CRC patients with high ERß expression had significantly higher levels of membrane-associated ß-catenin, cysteinyl leukotriene receptor 2 (CysLT2 R), and 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which have anti-tumour effects, but lower levels of nuclear ß-catenin, cysteinyl leukotriene receptor 1 (CysLT1 R), and cyclooxygenase-2 (COX-2), which have tumour-promoting effects. These interesting findings were further supported by two different publicly available CRC mRNA datasets that showed a significant positive correlation between ERß expression and 15-PGDH and CysLT2 R expression and a negative correlation between ERß expression and ß-catenin, CysLT1 R, and COX-2 expression. We next evaluated ERß expression in three different colon cancer mouse models; ERß expression was negatively correlated with tumourigenesis. Furthermore, treatment with the ERß-agonist ERB-041 reduced CysLT1 R, active ß-catenin, and COX-2 levels but increased phospho-ß-catenin, CysLT2 R, and 15-PGDH levels in HCT-116, Caco-2, and SW-480 colon cancer cells compared to vehicle-treated cells. Interestingly, ERB-041-treated cells showed significantly decreased migration, survival, and colonosphere formation and increased apoptotic activity, as indicated by increased CASPASE-3 and apoptotic blebs. Finally, patients with low ERß expression had significantly more distant metastasis at the time of diagnosis than patients with high ERß expression. Therefore, we studied the effects of ERB-041-treated colon cancer cells in a zebrafish xenograft model. We found significantly less distant metastasis of ERB-041-treated cells compared to vehicle-treated cells. These results further support ERß's anti-tumour role in CRC and the possible use of its agonist in CRC patients. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A mitotic CDK5-PP4 phospho-signaling cascade primes 53BP1 for DNA repair in G1.

Mitotic cells attenuate the DNA damage response (DDR) by phosphorylating 53BP1, a critical DDR mediator, to prevent its localization to damaged chromatin. Timely dephosphorylation of 53BP1 is critical for genome integrity, as premature recruitment of 53BP1 to DNA lesions impairs mitotic fidelity. Protein phosphatase 4 (PP4) dephosphorylates 53BP1 in late mitosis to allow its recruitment to DNA lesions in G1. How cells appropriately dephosphorylate 53BP1, thereby restoring DDR, is unclear. Here, we elucidate the underlying mechanism of kinetic control of 53BP1 dephosphorylation in mitosis. We demonstrate that CDK5, a kinase primarily functional in post-mitotic neurons, is active in late mitotic phases in non-neuronal cells and directly phosphorylates PP4R3ß, the PP4 regulatory subunit that recognizes 53BP1. Specific inhibition of CDK5 in mitosis abrogates PP4R3ß phosphorylation and abolishes its recognition and dephosphorylation of 53BP1, ultimately preventing the localization of 53BP1 to damaged chromatin. Our results establish CDK5 as a regulator of 53BP1 recruitment.

Montelukast, a CysLT1 receptor antagonist, reduces colon cancer stemness and tumor burden in a mouse xenograft model of human colon cancer.

Inflammation is implicated in the etiology of sporadic colon cancer (CC), which is one of the leading causes of cancer-related deaths worldwide. Here, we report that inhibition of the inflammatory receptor CysLT1 through its antagonist, montelukast, is beneficial in minimizing stemness in CC and thereby minimizing tumor growth in a mouse xenograft model of human colon cancer. Upon treatment with montelukast, colonospheres derived from HT-29 and SW-480 human colon cancer cells exhibited a significant phenotypic change coupled with the downregulation of mRNA and protein expression of cancer stem cell (CSC) markers ALDH1 and DCLK1. Moreover, montelukast reduced the size of HT-29 cell-derived tumors in mice. The reduction in tumor size was associated with decreased levels of ALDH1A1, DCLK1, BCL2 mRNA and macrophage infiltration into the tumor tissue. Interestingly, this treatment elevated levels of the tumor suppressor 15-PGDH while reducing COX-2 expression. Our data highlight the association of CysLT1R with CSCs and demonstrate that inhibition of CysLT1R could prove beneficial in minimizing CSC-induced tumor growth. This work advances the notion that targeting CSCs is a promising approach to improve outcomes in those afflicted with colon cancer.

Regulation of inside-out ß1-integrin activation by CDCP1.

Tumor metastasis depends on the dynamic regulation of cell adhesion through ß1-integrin. The Cub-Domain Containing Protein-1, CDCP1, is a transmembrane glycoprotein which regulates cell adhesion. Overexpression and loss of CDCP1 have been observed in the same cancer types to promote metastatic progression. Here, we demonstrate reduced CDCP1 expression in high-grade, primary prostate cancers, circulating tumor cells and tumor metastases of patients with castrate-resistant prostate cancer. CDCP1 is expressed in epithelial and not mesenchymal cells, and its cell surface and mRNA expression declines upon stimulation with TGFß1 and epithelial-to-mesenchymal transition. Silencing of CDCP1 in DU145 and PC3 cells resulted in 3.4-fold higher proliferation of non-adherent cells and 4.4-fold greater anchorage independent growth. CDCP1-silenced tumors grew in 100% of mice, compared to 30% growth of CDCP1-expressing tumors. After CDCP1 silencing, cell adhesion and migration diminished 2.1-fold, caused by loss of inside-out activation of ß1-integrin. We determined that the loss of CDCP1 reduces CDK5 kinase activity due to the phosphorylation of its regulatory subunit, CDK5R1/p35, by c-SRC on Y234. This generates a binding site for the C2 domain of PKCδ, which in turn phosphorylates CDK5 on T77. The resulting dissociation of the CDK5R1/CDK5 complex abolishes the activity of CDK5. Mutations of CDK5-T77 and CDK5R1-Y234 phosphorylation sites re-establish the CDK5/CDKR1 complex and the inside-out activity of ß1-integrin. Altogether, we discovered a new mechanism of regulation of CDK5 through loss of CDCP1, which dynamically regulates ß1-integrin in non-adherent cells and which may promote vascular dissemination in patients with advanced prostate cancer.

Metallic gold and bioactive quinacrine hybrid nanoparticles inhibit oral cancer stem cell and angiogenesis by deregulating inflammatory cytokines in p53 dependent manner.

Complete eradication of aggressive oral cancer remains a challenge due to the presence of CSCs. They resist conventional chemotherapeutic agents due to their self-renewal, drug efflux, and efficient DNA repair capacity. Here, we formulated a hybrid-nanoparticle (QAuNP) using quinacrine and gold and characterized/investigated its anti-angiogenic and anti-metastatic effect on OSCC-CSCs. QAuNP significantly inhibited cellular proliferation, caused apoptosis in vitro, and disrupted angiogenesis in vivo and tumor regression in xenograft mice model. It not only inhibited crucial angiogenic markers Ang-1, Ang-2 and VEGF but also depleted MMP-2 in H-357-PEMT cells in a p53 and p21-dependent manner. QAuNP also increased the ROS and NO generation in OSCC-CSCs and reduced the mitochondrial membrane potential. It altered the level of inflammatory cytokines IL-6, IL-1ß, TNF-α and metastasis-associated markers (CD-44, CD-133) in H-357-PEMT and CM-treated endothelial cells (HUVEC) in p53/p21-dependent manner. Therefore, QAuNP will be a useful therapeutic agent against metastatic OSCC.

A potential anti-tumor effect of leukotriene C4 through the induction of 15-hydroxyprostaglandin dehydrogenase expression in colon cancer cells.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Cyclooxygenase-2, which plays a key role in the biosynthesis of prostaglandin E2 (PGE2), is often up-regulated in CRC and in other types of cancer. PGE2 induces angiogenesis and tumor cell survival, proliferation and migration. The tumor suppressor 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme in PGE2 catabolism, converting it into its inactive metabolite 15-keto-PGE2, and is often down-regulated in cancer. Interestingly, CRC patients expressing high levels of the cysteinyl leukotriene 2 (CysLT2) receptor have a good prognosis; therefore, we investigated a potential link between CysLT2 signaling and the tumor suppressor 15-PGDH in colon cancer cells.We observed a significant up-regulation of 15-PGDH after treatment with LTC4, a CysLT2 ligand, in colon cancer cells at both the mRNA and protein levels, which could be reduced by a CysLT2 antagonist or a JNK inhibitor. LTC4 induced 15-PGDH promoter activity via JNK/AP-1 phosphorylation. Furthermore, we also observed that LTC4, via the CysLT2/JNK signaling pathway, increased the expression of the differentiation markers sucrase-isomaltase and mucin-2 in colon cancer cells and that down-regulation of 15-PGDH totally abolished the observed increase in these markers.In conclusion, the restoration of 15-PGDH expression through CysLT2 signaling promotes the differentiation of colon cancer cells, indicating an anti-tumor effect of CysLT2 signaling.

Nanoquinacrine induced apoptosis in cervical cancer stem cells through the inhibition of hedgehog-GLI1 cascade: Role of GLI-1.

To improve the pharmacokinetics and to study the anti-cervical cancer and anti-stem cells (CSCs) mechanism of Quinacrine (QC), a spherical nano particle of QC (i.e. NQC) was prepared and characterized. QC and NQC showed higher cytotoxicity in multiple cancer cells than the normal epithelial cells. NQC exhibited more toxicity in cervical cancer cells and its CSCs than QC. A dose-dependent decreased expression of Hedgehog-GLI (HH-GLI) components were noted in NQC treated HeLa cells and its CSCs. NQC increased the expressions of negatively regulated HH-GLI components (GSK3ß, PTEN) and caused apoptosis in CSCs. Reduction of GLI1 at mRNA and promoter level were noted after NQC exposure. The expressions of HH-GLI components, GLI1 promoter activity and apoptosis were unaltered in NQC treated GLI1-knockdown cells. In silico, cell based and in vitro reconstitution assay revealed that NQC inhibit HH-GLI cascade by binding to the consensus sequence (5'GACCACCCA3') of GLI1 in GLI-DNA complex through destabilizing DNA-GLI1 complex. NQC reduced the tumors size and proliferation marker Ki-67 in an in vivo xenograft mice model. Thus, NQC induced apoptosis in cancers through inhibition of HH-GLI cascade by GLI1. Detail interaction of QC-DNA-GLI complex can pave path for anticancer drug design.

Chk1 inhibitor synergizes quinacrine mediated apoptosis in breast cancer cells by compromising the base excision repair cascade.

Quinacrine (QC) causes apoptosis in breast cancer cells by induction of DNA damage, arrest of cells in S-phase, and by topoisomerase inhibition. Here, we show that QC-mediated apoptosis is not only due to increased DNA damage but also by compromising cell cycle checkpoints and base excision repair (BER) capacity in breast cancer cells. QC decreased CHK1, CDKs (CDC2, MDM2, CDC6), cyclins (B1, E1) and CDC25-A in a dose dependent manner. The expression of basal ATR remains unaltered but pATR (Ser-428) increased after QC treatment. A CHK1 inhibitor, SB218078, was also tested alone and in combination with QC. Like QC, SB218078 caused apoptosis by DNA damage and S-phase arrest. The combination of QC and SB218078 increased apoptosis by blocking the cell cycle in G2/M, which caused a mitotic catastrophe, and induced DNA damage at a higher level in comparison to individual compound treatments. Both drugs individually or in combination decreased the levels of replication protein A (RPA). Measurement of the expression of BER (SP- and LP-BER) proteins and direct in vivo BER activity revealed that the QC/SB218078 combination caused apoptosis in cancer cells by disrupting the induction of BER, which represents a novel means of potentially treating breast cancer.