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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.

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.

WNT5A is a well-known mediator of melanoma cell invasion and metastasis via its ability to activate protein kinase C (PKC), which is monitored by phosphorylation of the endogenous PKC substrate myristoylated alanine-rich c-kinase substrate (MARCKS). However, a possible direct contribution of MARCKS in WNT5A-mediated melanoma cell invasion has not been investigated. Analyses of melanoma patient databases suggested that similar to WNT5A expression, MARCKS expression appears to be associated with increased metastasis. A relationship between the two is suggested by the findings that recombinant WNT5A (rWNT5A) induces both increased expression and phosphorylation of MARCKS, whereas WNT5A silencing does the opposite. Moreover, WNT5A-induced invasion of melanoma cells was blocked by siRNA targeting MARCKS, indicating a crucial role of MARCKS expression and/or its phosphorylation. Next, we employed a peptide inhibitor of MARCKS phosphorylation that did not affect MARCKS expression and found that it abolished WNT5A-induced melanoma cell invasion. Similarly, rWNT5A induced the accumulation of phosphorylated MARCKS in membrane protrusions at the leading edge of melanoma cells. Our results demonstrate that WNT5A-induced phosphorylation of MARCKS is not only an indicator of PKC activity but also a crucial regulator of the metastatic behavior of melanoma and therefore an attractive future antimetastatic target in melanoma patients.

Increased expression and signalling of WNT5A and interleukin-6 (IL-6) have both been shown to promote melanoma progression. Here, we investigated the proposed existence of a WNT5A-IL-6 positive feedback loop that drives melanoma migration and invasion. First, the HOPP algorithm revealed that the invasive phenotype of cultured melanoma cells was significantly correlated with increased expression of WNT5A or IL-6. In three invasive melanoma cell lines, endogenous WNT5A protein expression was related to IL-6 protein secretion. Knockdown with anti-IL-6 siRNAs or treating WM852 melanoma cells with a neutralising anti-IL-6 antibody reduced WNT5A protein expression. Conversely, the silencing of WNT5A expression by WNT5A siRNAs or treating WM852 melanoma cells with Box5 (a WNT5A antagonist) significantly reduced IL-6 secretion. Interestingly, these effects occurred at the protein level but not at the transcriptional levels. Functionally, we demonstrated that combined siRNA knockdown of WNT5A and IL-6 expression or the simultaneous inhibition of WNT5A and IL-6 signalling inhibited melanoma cell invasion more effectively than suppressing each factor individually. Together, our results demonstrate that WNT5A and IL-6 are connected through a positive feedback loop in melanoma cells and that the combined targeting of both molecules could serve as an effective therapeutic means to reduce melanoma metastasis.

Despite the clinical development of novel adjuvant and neoadjuvant chemotherapeutic drugs, metastatic breast cancer is one of the leading causes of cancer-related death among women. The present review focuses on the relevance, mechanisms, and therapeutic potential of targeting WNT5A as a future anti-metastatic treatment strategy for breast cancer patients by restoring WNT5A signaling as an innovative therapeutic option. WNT5A is an auto- and paracrine β-catenin-independent ligand that has been shown to induce tumor suppression as well as oncogenic signaling, depending upon cancer type. In breast cancer patients, WNT5A protein expression has been observed to be significantly reduced in between 45 and 75% of the cases and associated with early relapse and reduced disease-free survival. WNT5A triggers various downstream signaling pathways in breast cancer that primarily affect tumor cell migration and invasion. The accumulated in vitro results reveal that treatment of WNT5A-negative breast cancer cells with recombinant WNT5A caused different tumor-suppressive responses and in particular it impaired migration and invasion. The anti-migratory/invasive and anti-metastatic effects of reconstituting WNT5A signaling by the small WNT5A mimicking peptide Foxy5 form the basis for two successful clinical phase 1-studies aiming at determining safety and pharmacokinetics as well as defining dose-level for a subsequent phase 2-study. We conclude that re-installation of WNT5A signaling is an attractive and promising anti-metastatic therapeutic approach for future treatment of WNT5A-negative breast cancer patients.

In recent years, scientists have advocated the use of targeted therapies in the form of drugs that modulate genes and proteins that are directly associated with cancer progression and metastasis. Malignant melanoma is a dreadful cancer type that has been associated with the rapid dissemination of primary tumors to multiple sites, including bone, brain, liver and lungs. The discovery that approximately 40%-50% of malignant melanomas contain a mutation in BRAF at codon 600 gave scientists a new approach to tackle this disease. However, clinical studies on patients have shown that although BRAFi (BRAF inhibitors) trigger early anti-tumor responses, the majority of patients later develop resistance to the therapy. Recent studies have shown that WNT5A plays a key role in enhancing the resistance of melanoma cells to BRAFi. The focus of the current review will be on melanoma development, signaling pathways important to acquired resistance to BRAFi, and why WNT5A inhibitors are attractive candidates to be included in combinatorial therapies for melanoma.

Pentacyclic Triterpenoids (PTs) and their analogues as well as derivatives are emerging as important drug leads for various diseases. They act through a variety of mechanisms and a majority of them inhibit the nuclear factor kappa-beta (NF-κB) signaling pathway. In this study, we examined the effects of the naturally occurring PTs on IκB kinase-β (IKKβ), which has great scientific relevance in the NF-κB signaling pathway. On virtual screening, 109 PTs were screened through the PASS (prediction of activity spectra of substances) software for prediction of NF-κB inhibitory activity followed by docking on the NEMO/IKKβ association complex (PDB: 3BRV) and testing for compliance with the softened Lipinski's Rule of Five using Schrodinger (LLC, New York, USA). Out of the projected 45 druggable PTs, Corosolic Acid (CA), Asiatic Acid (AA) and Ursolic Acid (UA) were assayed for IKKβ kinase activity in the cell free medium. The UA exhibited a potent IKKβ inhibitory effect on the hotspot kinase assay with IC50 of 69 μM. Whereas, CA at 50 μM concentration markedly reduced the NF-κB luciferase activity and phospho-IKKβ protein expressions. The PTs tested, attenuated the expression of the NF-κB cascade proteins in the LPS-stimulated RAW 264.7 cells, prevented the phosphorylation of the IKKα/β and blocked the activation of the Interferon-gamma (IFN-γ). The results suggest that the IKKβ inhibition is the major mechanism of the PTs-induced NF-κB inhibition. PASS predictions along with in-silico docking against the NEMO/IKKβ can be successfully applied in the selection of the prospective NF-κB inhibitory downregulators of IKKβ phosphorylation.

Colorectal cancer (CRC) patients with APC mutations do not benefit from 5-FU therapy. It was reported that APC physically interacts with POLβ and FEN1, thus blocking LP-BER via APC's DNA repair inhibitory (DRI) domain in vitro. The aim of this study was to elucidate how APC status affects BER and the response of CRC to 5-FU. HCT-116, HT-29, and LOVO cells varying in APC status were treated with 5-FU to evaluate expression, repair, and survival responses. HCT-116 expresses wild-type APC; HT-29 expresses an APC mutant that contains DRI domain; LOVO expresses an APC mutant lacking DRI domain. 5-FU increased the expression of APC and decreased the expression of FEN1 in HCT-116 and HT-29 cells, which were sensitized to 5-FU when compared to LOVO cells. Knockdown of APC in HCT-116 rendered cells resistant to 5-FU, and FEN1 levels remained unchanged. Re-expression of full-length APC in LOVO cells caused sensitivity to 5-FU, and decreased expression of FEN1. These knockdown and addback studies confirmed that the DRI domain is necessary for the APC-mediated reduction in LP-BER and 5-FU. Modelling studies showed that 5-FU can interact with the DRI domain of APC via hydrogen bonding and hydrophobic interactions. 5-FU resistance in CRC occurs with mutations in APC that disrupt or eliminate the DRI domain's interaction with LP-BER. Understanding the type of APC mutation should better predict 5-FU resistance in CRC than simply characterizing APC status as wild-type or mutant.

Here, we report the p53 dependent mitochondria- mediated apoptotic mechanism of plant derived silver- nanoparticle (PD-AgNPs) in colorectal cancer cells (CRCs). PD-AgNPs was synthesized by reduction of AgNO3 with leaf extract of a medicinal plant periwinkle and characterized. Uptake of PD-AgNPs (ξ - 2.52±4.31 mV) in HCT116 cells was 3 fold higher in comparison to synthetic AgNPs (ξ + 2.293±5.1 mV). A dose dependent increase in ROS produc- tion, activated JNK and decreased mitochondrial membrane potential (MMP) were noted in HCT116 but not in HCT116 p53 �/� cells after PD-AgNP exposure. PD-AgNP-mediated apoptosis in CRCs is a p53 dependent process involving ROS and JNK cascade.

We previously reported that quinacrine (QC) has anticancer activity against breast cancer cells. Here, we examine the mechanism of action of QC and its ability to inhibit Wnt-TCF signaling in two independent breast cancer cell lines. QC altered Wnt-TCF signaling components by increasing the levels of adenomatous polyposis coli (APC), DAB2, GSK-3β and axin and decreasing the levels of β-catenin, p-GSK3β (ser 9) and CK1. QC also reduced the activity of the Wnt transcription factor TCF/LEF and its downstream targets cyclin D1 and c-MYC. Using a luciferase-based Wnt-TCF transcription factor assay, it was shown that APC levels were inversely associated with TCF/LEF activity. Induction of apoptosis and DNA damage was observed after treatment with QC, which was associated with increased expression of APC. The effects induced by QC depend on APC because the inhibition of Wnt-TCF signaling by QC is lost in APC-knockdown cells, and consequently, the extent of apoptosis and DNA damage caused by QC is reduced compared with parental cells. Because we previously showed that QC inhibits topoisomerase, we examined the effect of another topoisomerase inhibitor, etoposide, on Wnt signaling. Interestingly, etoposide treatment also reduced TCF/LEF activity, β-catenin and cyclin D1 levels commensurate with induction of DNA damage and apoptosis. Lycopene, a plant-derived antioxidant, synergistically increased QC activity and inhibited Wnt-TCF signaling in cancer cells without affecting the MCF-10A normal breast cell line. Collectively, the data suggest that QC-mediated Wnt-TCF signal inhibition depends on APC and that the addition of lycopene synergistically increases QC anticancer activity.

Most current anti-cancer therapeutic drugs are targeting the proliferation and/or survival of cancer cells while very few drugs are aimed at specifically targeting the dissemination process. Several reports have demonstrated that low-levels or lack of Wnt-5a protein expression in primary breast-, colon-, and prostate cancer tissues correlates with shortened patient recurrence-free survival and overall survival pointing to a biological role of Wnt5a signaling in the dissemination process of cancer cells. The Wnt5a ligand mediates its effects vid interaction with G-protein coupled Frizzled receptors and tyrosine-kinase coupled receptors such as ROR1 and ROR2. Therefore, we developed two peptides, one being a Wnt-5a agonist (Foxy-5) and one being a Wnt-5a antagonist (Box-5). The Foxy-5 is a formylated Wnt5a-derived hexapeptide that mimics the ability of the Wnt-5a molecule to impair cancer cell migration in vitro and significantly reduces the formation of distant metastases in vivo in mouse models of breast- and prostate cancer. In addition, a 4-week toxicology study in rats and dogs with a final dose well exceeding the dose previously used in the mouse models showed no drug-induced toxic reactions.Based on all these pre-clinical data we initiated a clinical phase 1 study with the primary objective to evaluate the safety and tolerability of Foxy-5. All eligible patients are pre-screened for Wnt-5a immunoreactivity in archival tumor tissue and from dose level 7 and onwards only patients with negative or low level Wnt5a expressing metastatic breast-, colon-, or prostate cancer are enrolled in the study. This study has currently recruited cohorts 1-7 without reaching MTD and the final recruitment for the last dose level (dose level 8) is ongoing. Clinical trial information: NCT02020291. This study will be finalized during early fall 2015 and followed by a phase 1b study which will continue dose escalation but with a specific focus on determining the Biological Active Dose (BAD) since Foxy-5 does not possess anti-proliferative activities and is therefore not expected to induce tumor regression. In contrast to breast, colon and prostate cancer, Wnt5a signaling promotes tumor progression in vitro in melanoma and gastric cancer. The antagonistic Wnt5a-derived peptide Box5 possesses the capacity to impair Wnt5a signaling and migration in melanoma cells. We are now performing additional pre-clinical experiments to enable us to test the in vivo effects of Box5 in a melanoma animal model. Future clinical development plans include a phase II study enrolling patients with stage III colorectal cancer and then add Foxy-5 to standard 5FU plus oxaliplatin adjuvant treatment. Citation Format: Tommy Andersson, Lena Axelsson, Purusottam Mohapatra, Chandra Prasad, Peter Grundtvig Soerensen, Morten Mau-Soerensen, Ulrik Lassen, Tine Molvadgaard, Ulla Buhl, Nils Brünner, Dorte Nielsen. Targeting the Wnt-5a signaling pathway as a novel anti-metastatic therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A116.

We previously reported that quinacrine (QC) has anticancer activity against breast cancer cells. Here, we report the mechanism of action of QC and its ability to inhibit Wnt-TCF signaling in two independent breast cancer cell lines. QC altered Wnt-TCF signaling components by increasing the levels of adenomatous polyposis coli (APC), DAB2, GSK-3β and axin and decreasing the levels of β-catenin, p-GSK3β (ser 9) and CK1. QC also reduced the activity of the Wnt transcription factor TCF/LEF and its downstream targets cyclin D1 and c-MYC. Using a luciferase-based Wnt-TCF transcription factor assay, it was shown that APC levels were inversely associated with TCF/LEF activity. Induction of apoptosis and DNA damage was observed after treatment with QC, which was associated with increased expression of APC. The effects induced by QC depend on APC because the inhibition of Wnt-TCF signalling by QC is lost in APC-knockdown cells, and consequently, the extent of apoptosis and DNA damage caused by QC was reduced compared with parental cells. Further, previously we have shown that QC inhibits topoisomerase, therefore we tested the effect of another topoisomerase inhibitor, etoposide, on Wnt signaling. Interestingly, etoposide treatment also reduced TCF/LEF activity, β-catenin and cyclin D1 levels commensurate with induction of DNA damage and apoptosis. Lycopene, a plant-derived antioxidant, synergistically increased QC activity and inhibited Wnt-TCF signaling in cancer cells without affecting the MCF-10A normal breast cell line. Collectively, the data suggests that QC-mediated Wnt-TCF signal inhibition depends on APC and that the addition of lycopene synergistically increases QC anticancer activity. Note: This abstract was not presented at the meeting. Citation Format: Ranjan Preet, Rajasubramaniam Shanmugam, Purusottam Mohapatra, Dipon Das, Shakti R. Satapathy, Michael D. Wyatt, Chanakya N. Kundu. Lycopene synergistically enhances quinacrine action to inhibit Wnt-TCF signaling in breast cancer cells through APC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2777. doi:10.1158/1538-7445.AM2014-2777

To study the mechanism of 5-fluorouracil (5-FU) resistance in colon cancer cells and to develop strategies for overcoming such resistance by combination treatment.We established and characterized a 5-FU resistance (5-FU-R) cell line derived from continuous exposure (25 μmol/L) to 5-FU for 20 wk in 5-FU sensitive HCT-116 cells. The proliferation and expression of different representative apoptosis and anti-apoptosis markers in 5-FU sensitive and 5-FU resistance cells were measured by the MTT assay and by Western blotting, respectively, after treatment with Resveratrol (Res) and/or 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU). Apoptosis and cell cycle arrest was measured by 4',6'-diamidino-2-phenylindole hydrochloride staining and fluorescence-activated cell sorting analysis, respectively. The extent of DNA damage was measured by the Comet assay. We measured the visible changes in the DNA damage/repair cascade by Western blotting.The widely used chemotherapeutic agents BCNU and Res decreased the growth of 5-FU sensitive HCT-116 cells in a dose dependent manner. Combined application of BCNU and Res caused more apoptosis in 5-FU sensitive cells in comparison to individual treatment. In addition, the combined application of BCNU and Res caused a significant decrease of major DNA base excision repair components in 5-FU sensitive cells. We established a 5-FU resistance cell line (5-FU-R) from 5-FU-sensitive HCT-116 (mismatch repair deficient) cells that was not resistant to other chemotherapeutic agents (e.g., BCNU, Res) except 5-FU. The 5-FU resistance of 5-FU-R cells was assessed by exposure to increasing concentrations of 5-FU followed by the MTT assay. There was no significant cell death noted in 5-FU-R cells in comparison to 5-FU sensitive cells after 5-FU treatment. This resistant cell line overexpressed anti-apoptotic [e.g., AKT, nuclear factor κB, FLICE-like inhibitory protein), DNA repair (e.g., DNA polymerase beta (POL-β), DNA polymerase eta (POLH), protein Flap endonuclease 1 (FEN1), DNA damage-binding protein 2 (DDB2)] and 5-FU-resistance proteins (thymidylate synthase) but under expressed pro-apoptotic proteins (e.g., DAB2, CK1) in comparison to the parental cells. Increased genotoxicity and apoptosis were observed in resistant cells after combined application of BCNU and Res in comparison to untreated or parental cells. BCNU increased the sensitivity to Res of 5-FU resistant cells compared with parental cells. Fifty percent cell death were noted in parental cells when 18 μmol/L of Res was associated with fixed concentration (20 μmol/L) of BCNU, but a much lower concentration of Res (8 μmol/L) was needed to achieve the same effect in 5-FU resistant cells. Interestingly, increased levels of adenomatous polyposis coli and decreased levels POL-β, POLH, FEN1 and DDB2 were noted after the same combined treatment in resistant cells.BCNU combined with Res exerts a synergistic effect that may prove useful for the treatment of colon cancer and to overcome drug resistance.