Chandra Prakash Prasad

Interleukin-6 drives melanoma cell motility through p38α-MAPK-dependent up-regulation of WNT5A expression

Extensive research has demonstrated a tumor‐promoting role of increased WNT5A expression in malignant melanoma. However, very little light has been shed upon how WNT5A expression is up‐regulated in melanoma. A potential regulator of WNT5A expression is the pro‐inflammatory cytokine Interleukin (IL)‐6, which shares the ability of WNT5A to increase melanoma cell invasion. Here, we investigate whether IL‐6 can promote melanoma cell motility through an increased expression of WNT5A. We clearly demonstrate that the WNT5A‐antagonistic peptide Box5 could inhibit IL‐6‐induced melanoma cell migration and invasion. Furthermore, IL‐6 stimulation of the human melanoma cell lines HTB63 and A375 increased the expression of WNT5A in a dose‐dependent manner. To identify the signaling mechanism responsible for this up‐regulation, we explored the involvement of the three main signals induced by IL‐6; STAT3, Akt and ERK 1/2. Of these, only STAT3 was activated by IL‐6 in the melanoma cell lines tested. However, the STAT3 inhibitor S3I‐201 failed to inhibit IL‐6‐induced WNT5A up‐regulation in HTB63 and A375 cells. Nor did STAT3 siRNA silencing affect the expression of WNT5A. In search of an alternative signaling mechanism, we detected IL‐6‐induced activation of p38‐MAPK in HTB63 and A375 cells. The p38‐MAPK inhibitor SB203580 abolished the IL‐6‐induced WNT5A up‐regulation and blocked IL‐6‐induced melanoma cell invasion. The latter effect could be rescued by the addition of recombinant WNT5A. Notably, immunoprecipitation analysis revealed that only the p38α‐MAPK isoform was activated by IL‐6, and subsequent siRNA silencing of p38α‐MAPK abolished the IL‐6‐induced up‐regulation of WNT5A. Taken together, we demonstrate a novel link between the two melanoma pro‐metastatic agents IL‐6 and WNT5A explaining how IL‐6 can increase melanoma cell invasion and thus promote the metastatic process. This finding provides a basis for future therapeutic intervention of melanoma progression.

WNT-5A triggers Cdc42 activation leading to an ERK1/2 dependent decrease in MMP9 activity and invasive migration of breast cancer cells.

An important role for WNT‐5A is implicated in a variety of tumors, including breast carcinoma. We previously showed that WNT‐5A signaling inhibits migration and metastasis of breast cancer cells, and that patients with primary breast cancer in which WNT‐5A was expressed have a better prognosis. Despite the fact that RhoGTPase Cdc42 is commonly associated with increased cell migration, we here show that recombinant WNT‐5A activates the Cdc42 in breast cancer cells (lines MDA‐MB468 and MDA‐MB231) in a time‐dependent manner. Activation of Cdc42 was also observed in MDA‐MB468 cells that were stably transfected with a WNT‐5A plasmid (MDA‐MB468‐5A). In all situations, increased Cdc42 activity was accompanied by decreased migration and invasion of the breast cancer cells. To explore these findings further we also investigated the effect of WNT‐5A signaling on ERK1/2 activity. Apart from an initial Ca2+‐dependent rWNT‐5A‐induced activation of ERK1/2, Cdc42 activity was inversely correlated with ERK1/2 activity in both rWNT‐5A‐stimulated parental MDA‐MB468 and MDA‐MB468‐5A cells. We also demonstrated increased ERK1/2 activity in MDA‐MB468‐5A cells following siRNA knockdown of Cdc42. Consistent with these results, breast cancer cells transfected with constitutively active Cdc42 exhibited reduced ERK1/2 activity, migration and invasion, whereas cells transfected with dominant negative Cdc42 had increased ERK1/2 activity in response to rWNT‐5A. To gain information on how ERK1/2 can mediate its effect on breast cancer cell migration and invasion, we next investigated and demonstrated that WNT‐5A signaling and constitutively active Cdc42 both decreased matrix metalloproteinase 9 (MMP9) activity. These data indicate an essential role of Cdc42 and ERK1/2 signaling and MMP9 activity in WNT‐5A‐impaired breast cancer cells.

Non-canonical WNT5A signaling up-regulates the expression of the tumor suppressor 15-PGDH and induces differentiation of colon cancer cells

The tumor suppressor 15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) is the key enzyme in prostaglandin E2 catabolism and is down‐regulated in colorectal cancer (CRC) tissue. Canonical Wnt signaling is frequently elevated in colon cancers and has been shown to down‐regulate 15‐PGDH expression. Therefore, we have in the current study investigated if the non‐canonical ligand WNT5A relates to increased expression of 15‐PGDH in colon cancer cells. In the same cohort of patients, we demonstrated a parallel and significant loss of 15‐PGDH and WNT5A protein expression in CRC tissues compared with matched normal colon tissues. Furthermore, patients with low 15‐PGDH/WNT5A expression in their tumors showed reduced survival compared with patients with high 15‐PGDH/WNT5A expression. To investigate if WNT5A signaling directly affects 15‐PGDH expression, we performed in vitro analyses of colon cancer cells (HT‐29 and Caco‐2). Both cell lines, when treated with recombinant WNT5A (rWNT5A) or Foxy‐5, a WNT5A‐mimicking peptide, responded by increasing their expression of 15‐PGDH mRNA and protein. Our investigations showed that rWNT5A and Foxy‐5 induced this increased expression of 15‐PGDH through reduced β‐catenin signaling as well as increased JNK/AP‐1 signaling in colon cancer cells. WNT5A signaling also induced increased 15‐PGDH expression in a breast cancer cell line both in vitro and in vivo. In agreement, WNT5A signaling also increased the expression of the differentiation markers sucrose‐isomaltase and mucin‐2 in colon cancer cells. Our results show that WNT5A signaling regulates 15‐PGDH expression, thus uncovering a novel mechanism by which WNT5A acts as a tumor suppressor and suggests that increased 15‐PGDH expression could be used as an indicator of a positive response to Foxy‐5 in patients treated with this WNT5A agonist.

WNT5A signaling impairs breast cancer cell migration and invasion via mechanisms independent of the epithelial-mesenchymal transition

BackgroundWNT5A (-/-) mammary tissue has been shown to exhibit increased ductal elongation, suggesting elevated mammary cell migration. Increased epithelial cell migration/invasion has often but not always been linked to the epithelial-mesenchymal transition (EMT). In the current study, we investigated the loss of WNT5A in HB2 human mammary epithelial cells and hypothesized that this loss increased their invasion via the EMT. Based on these results, we postulated that suppression of breast cancer cell migration and invasion by WNT5A is due to EMT reversal.MethodsWNT5A was transiently knocked down using specific siRNAs, whereas WNT5A signaling was induced in MDA-MB468 and MDA-MB231 breast cancer cells by stably transfecting cells with WNT5A or treating them with recombinant WNT5A (rWNT5A). Changes in EMT markers, CD44, pAKT and AKT expression were assessed using Western blotting and immunofluorescence. The physiological relevance of altered WNT5A signaling was assessed using migration and invasion assays.ResultsWNT5A knockdown in HB2 mammary epithelial cells resulted in EMT-like changes and increased invasiveness, and these changes were partially reversed by the addition of rWNT5A. These data suggest that WNT5A might inhibit breast cancer cell migration and invasion by a similar EMT reversal. Contrary to our expectations, we did not observe any changes in the EMT status of breast cancer cells, either after treatment with rWNT5A or stable transfection with a WNT5A plasmid, despite the parallel WNT5A-induced inhibition of migration and invasion. Instead, we found that WNT5A signaling impaired CD44 expression and its downstream signaling via AKT. Moreover, knocking down CD44 in breast cancer cells using siRNA impaired cell migration and invasion.ConclusionsWNT5A bi-directionally regulates EMT in mammary epithelial cells, thereby affecting their migration and invasion. However, the ability of WNT5A to inhibit breast cancer cell migration and invasion is an EMT-independent mechanism that, at least in part, can be explained by decreased CD44 expression.

Therapy for BRAFi-Resistant Melanomas: Is WNT5A the Answer?

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.

Treatment with the WNT5A-mimicking peptide Foxy-5 effectively reduces the metastatic spread of WNT5A-low prostate cancer cells in an orthotopic mouse model

Prostate cancer patients with high WNT5A expression in their tumors have been shown to have more favorable prognosis than those with low WNT5A expression. This suggests that reconstitution of Wnt5a in low WNT5A-expressing tumors might be an attractive therapeutic approach. To explore this idea, we have in the present study used Foxy-5, a WNT5A mimicking peptide, to investigate its impact on primary tumor and metastasis in vivo and on prostate cancer cell viability, apoptosis and invasion in vitro. We used an in vivo orthotopic xenograft mouse model with metastatic luciferase-labeled WNT5A-low DU145 cells and metastatic luciferase-labeled WNT5A-high PC3prostate cancer cells. We provide here the first evidence that Foxy-5 significantly inhibits the initial metastatic dissemination of tumor cells to regional and distal lymph nodes by 90% and 75%, respectively. Importantly, this effect was seen only with the WNT5A-low DU145 cells and not with the WNT5A-high PC3 cells. The inhibiting effect in the DU145-based model occurred despite the fact that no effects were observed on primary tumor growth, apoptosis or proliferation. These findings are consistent with and supported by the in vitro data, where Foxy-5 specifically targets invasion without affecting apoptosis or viability of WNT5A-low prostate cancer cells. To conclude, our data indicate that the WNT5A-mimicking peptide Foxy-5, which has been recently used in a phase 1 clinical trial, is an attractive candidate for complimentary anti-metastatic treatment of prostate cancer patients with tumors exhibiting absent or low WNT5A expression.

Dual mechanisms of action of the RNA-binding protein human antigen R explains its regulatory effect on melanoma cell migration

Overexpression of wingless-type MMTV integration site family 5A (WNT5A) plays a significant role in melanoma cancer progression; however, the mechanism(s) involved remains unknown. In breast cancer, the human antigen R (HuR) has been implicated in the regulation of WNT5A expression. Here, we demonstrate that endogenous expression of WNT5A correlates with levels of active HuR in HTB63 and WM852 melanoma cells and that HuR binds to WNT5A messenger RNA in both cell lines. Although the HuR inhibitor MS-444 significantly impaired migration in both melanoma cell lines, it reduced WNT5A expression only in HTB63 cells, as did small interfering RNA knockdown of HuR. Consistent with this finding, MS-444-induced inhibition of HTB63 cell migration was restored by the addition of recombinant WNT5A, whereas MS-444-induced inhibition of WM852 cell migration was restored by the addition of recombinant matrix metalloproteinase-9, another HuR-regulated protein. Clearly, HuR positively regulates melanoma cell migration via at least 2 distinct mechanisms making HuR an attractive therapeutic target for halting melanoma dissemination.

Reduced production and uptake of lactate are essential for the ability of WNT5A signaling to inhibit breast cancer cell migration and invasion

Here we investigated the impact of WNT5A signaling on aerobic glycolysis and evaluated its effects on breast cancer cell migration/invasion. WNT5A signaling reduced migration and lactate production and caused selective down-regulation of the glycolytic enzyme phosphofructokinase platelet-type (PFKP). These events occurred in parallel with a WNT5A-induced inhibition of β-catenin signaling. Support for essential involvement of β-catenin and PFKP in lactate production and migration/invasion was obtained by siRNA knockdown of their expression. To also explore the effect of non-tumor cell-derived lactate, we added exogenous lactate to the cells and noted an increase in migration that was significantly impaired by recombinant WNT5A in parallel with a down-regulation of the lactate transporter monocarboxylate transporter 1 (MCT1). Interestingly enough, the drug-candidate Foxy5 (WNT5A-mimic hexapeptide) also inhibited breast cancer cell migration in the presence of exogenous lactate, suggesting a therapeutic potential for Foxy5 in managing breast tumors with high glycolytic activity. Overall, we demonstrated that WNT5A signaling (via a β-catenin-PFKP axis) reduces lactate production and lowers the expression of MCT1, a carrier mediating the uptake of lactate from the tumor microenvironment. These effects of WNT5A are essential for its ability to impair breast cancer migration/invasion even in an environment with elevated lactate levels.Here we investigated the impact of WNT5A signaling on aerobic glycolysis and evaluated its effects on breast cancer cell migration/invasion. WNT5A signaling reduced migration and lactate production and caused selective down-regulation of the glycolytic enzyme phosphofructokinase platelet-type (PFKP). These events occurred in parallel with a WNT5A-induced inhibition of β-catenin signaling. Support for essential involvement of β-catenin and PFKP in lactate production and migration/invasion was obtained by siRNA knockdown of their expression. To also explore the effect of non-tumor cell-derived lactate, we added exogenous lactate to the cells and noted an increase in migration that was significantly impaired by recombinant WNT5A in parallel with a down-regulation of the lactate transporter monocarboxylate transporter 1 (MCT1). Interestingly enough, the drug-candidate Foxy5 (WNT5A-mimic hexapeptide) also inhibited breast cancer cell migration in the presence of exogenous lactate, suggesting a therapeutic potential for Foxy5 in managing breast tumors with high glycolytic activity. Overall, we demonstrated that WNT5A signaling (via a β-catenin-PFKP axis) reduces lactate production and lowers the expression of MCT1, a carrier mediating the uptake of lactate from the tumor microenvironment. These effects of WNT5A are essential for its ability to impair breast cancer migration/invasion even in an environment with elevated lactate levels.

Demonstration of a WNT5A-IL-6 positive feedback loop in melanoma cells : Dual interference of this loop more effectively impairs melanoma cell invasion

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.

Abstract A116: Targeting the Wnt-5a signaling pathway as a novel anti-metastatic therapy

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 Brunner, 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.