Summary Stem-like glioma cells reside within a perivascular niche and display hallmark radiation resistance. Understanding of the mechanisms underlying these properties will be vital for the development of effective therapies. Here we show that the stem cell marker CD44 promotes cancer stem cell phenotypes and radiation resistance. In a mouse model of glioma, Cd44−/− and Cd44+/− animals showed improved survival compared to controls. The CD44 ligand Osteopontin shared a perivascular expression pattern with CD44 and promoted glioma stem cell-like phenotypes. These effects were mediated via the γ-secretase regulated intracellular domain of CD44, which promoted aggressive glioma growth in vivo and stem cell-like phenotypes via CBP/p300-dependent enhancement of HIF-2α activity. In human glioblastoma multiforme, expression of CD44 correlated with hypoxia-induced gene signatures and poor survival. Together, these data suggest that in the glioma perivascular niche, Osteopontin promotes stem cell-like properties and radiation resistance in adjacent tumor cells via activation of CD44 signaling.

BackgroundWnt proteins are important for developmental processes and certain diseases. WNT5A is a non-canonical Wnt protein that previously has been shown to play a role in the progression of malignant melanoma. High expression of WNT5A in melanoma tumors correlates to formation of distant metastasis and poor prognosis. This has partly been described by the findings that WNT5A expression in melanoma cell lines increases migration and invasion.MethodsMalignant melanoma cell lines were treated with rWNT5A or WNT5A siRNA, and mRNA versus protein levels of soluble mediators were measured using RT-PCR, cytokine bead array and ELISA. The induced signaling pathways were analyzed using inhibitors, Rho-GTPase pull down assays and western blot. Ultracentrifugation and electron microscopy was used to analyze microvesicles. Gene expression microarray data obtained from primary malignant melanomas was used to verify our data.ResultsWe show that WNT5A signaling induces a Ca2+-dependent release of exosomes containing the immunomodulatory and pro-angiogenic proteins IL-6, VEGF and MMP2 in melanoma cells. The process was independent of the transcriptional machinery and depletion of WNT5A reduced the levels of the exosome-derived proteins. The WNT5A induced exosomal secretion was neither affected by Tetanus toxin nor Brefeldin A, but was blocked by the calcium chelator Bapta, inhibited by a dominant negative version of the small Rho-GTPase Cdc42 and was accompanied by cytoskeletal reorganization. Co-cultures of melanoma/endothelial cells showed that depletion of WNT5A in melanoma cells decreased endothelial cell branching, while stimulation of endothelial cells with isolated rWNT5A-induced melanoma exosomes increased endothelial cell branching in vitro. Finally, gene expression data analysis of primary malignant melanomas revealed a correlation between WNT5A expression and the angiogenesis marker ESAM.ConclusionsThese data indicate that WNT5A has a broader function on tumor progression and metastatic spread than previously known; by inducing exosome-release of immunomodulatory and pro-angiogenic factors that enhance the immunosuppressive and angiogenic capacity of the tumors thus rendering them more aggressive and more prone to metastasize.

A well-orchestrated inflammatory reaction involves the induction of effector functions and, at a later stage, an active downregulation of this potentially harmful process. In this study we show that under proinflammatory conditions the noncanonical Wnt protein, Wnt5a, induces immunosuppressive macrophages. The suppressive phenotype induced by Wnt5a is associated with induction of IL-10 and inhibition of the classical TLR4-NF-κB signaling. Interestingly, this phenotype closely resembles that observed in reprogrammed monocytes in sepsis patients. The Wnt5a-induced feedback inhibition is active both during in vitro LPS stimulation of macrophages and in patients with sepsis caused by LPS-containing, Gram-negative bacteria. Furthermore, using breast cancer patient tissue microarrays, we find a strong correlation between the expression of Wnt5a in malignant epithelial cells and the frequency of CD163+ anti-inflammatory tumor-associated macrophages. In conclusion, our data point out Wnt5a as a potential target for an efficient therapeutic modality in severe human diseases as diverse as sepsis and malignancy.

WNT5A has been identified as an important ligand in the malignant progression of a number of tumours. Although WNT5A signalling is often altered in cancer, the ligand’s role as either a tumour suppressor or oncogene varies between tumour types and is a contemporary issue for investigators of β-catenin-independent WNT signalling in oncology. Here, we report that one of the initial effects of active WNT5A signalling in malignant melanoma cells is an alteration in cellular energy metabolism and specifically an increase in aerobic glycolysis. This was found to be at least in part due to an increase in active Akt signalling and lactate dehydrogenase (LDH) activity. The clinical relevance of these findings was strengthened by a strong correlation (P < 0.001) between the expression of WNT5A and LDH isoform V in a cohort of melanocytic neoplasms. We also found effects of WNT5A on energy metabolism in breast cancer cells, but rather than promoting aerobic glycolysis as it does in melanoma, WNT5A signalling increased oxidative phosphorylation rates in breast cancer cells. These findings support a new role for WNT5A in the metabolic reprogramming of cancer cells that is a context- dependent event.

BackgroundWnt signaling is important in development and can also contribute to the initiation and progression of cancer. The Secreted Frizzled Related Proteins (SFRPs) constitute a family of Wnt modulators, crucial for controlling Wnt signaling. Here we investigate the expression and role of SFRP3 in melanoma.Methodology/Principal FindingsWe show that SFRP3 mRNA is down-regulated in malignant melanoma tumors as compared to normal/benign tissue. Furthermore, we found that SFRP3 expression was lost in the malignant melanoma cell lines, A2058, HTB63 and A375, but not in the non-transformed melanocyte cell line, Hermes 3A. Methylated CpG rich areas were detected in the SFRP3 gene in melanoma cell lines and their SFRP3 expression could be restored using the demethylating agent, 5′aza-deoxycytidine. Addition of recombinant SFRP3 to melanoma cells had no effect on viable cell numbers, but decreased cell migration and invasion. Wnt5a signaling has been shown to increase the migration and invasion of malignant melanoma cells, and high expression of Wnt5a in melanoma tumors has been connected to a poor prognosis. We found that recombinant SFRP3 could inhibit Wnt5a signaling, and that it inhibited melanoma cell migration and invasion in a Wnt5a-dependent manner.Conclusion/SignificanceWe conclude that SFRP3 functions as a melanoma migration and invasion suppressor by interfering with Wnt5a signaling.

One third of all breast cancers are estrogen receptor alpha (ERα) negative, carry a poor overall prognosis, and do not respond well to currently available endocrine therapies. New treatment strategies are therefore required. Loss of Wnt-5a has previously been correlated with loss of ERα in clinical breast cancer samples, and we sought to investigate this association further. Three breast cancer cell lines (MDA-MB-231, MDA-MB-468, and 4T1) lacking expression of ERα and Wnt-5a, and one breast cancer cell line (T47D) expressing both proteins were used in this study. Wnt-5a signaling was generated in ERα-negative cell lines via stimulation with either recombinant Wnt-5a protein or a Wnt-5a-derived hexapeptide (Foxy-5) possessing Wnt-5a signaling properties. ERα expression was restored at both mRNA and protein level, after treatment with recombinant Wnt-5a or Foxy-5. This restoration of expression occurred in parallel with a reduction in methylation of the ERα promoter. Up-regulated ERα could be activated, initiate transcription of progesterone receptor and pS2, and activate an estrogen response element reporter construct. Significantly, breast cancer cells re-expressing ERα responded to treatment with the selective estrogen receptor modulator tamoxifen, as measured by induction of apoptosis and cell growth inhibition. Finally, Foxy-5 also increased ERα expression in an in vivo model of ERα-negative breast cancer. This represents the first evidence that Wnt-5a signaling acts to re-establish ERα expression in ERα-negative breast cancer cells. Our data suggest that combinatorial therapy with Foxy-5 and tamoxifen should be considered as a future treatment possibility for ERα-negative breast cancer patients.

Despite improvements in detection and treatment, breast cancer remains the most common female cancer worldwide, and metastatic associated mortality is a significant public health issue. Patients with tumors negative for estrogen receptor (ERalpha), have a particularly poor prognosis, partly due to their inability to respond to current endocrine treaments. Expression of Wnt-5a has been associated with prolonged recurrence free survivial in clinical material, and Wnt-5a also inhibits migration and invasion of breast cancer cell lines. Loss of Wnt-5a is associated with loss of ERalpha in clinical breast cancer material, and Wnt-5a signaling upregulates ERalpha in ERalpha negative breast cancer cell lines. A Wnt-5a derived hexapeptide, Foxy-5, has been developed and like Wnt-5a, increases adhesion and inhibits migration of breast cancer cells. Furthermore, Foxy-5 significantly reduced liver and lung metastases in a murine ERalpha negative breast cancer model. Foxy-5 also upregulated ERalpha in this in vivo model and most significantly, in vitro rendered cells responsive to the selective estrogen receptor modulator, Tamoxifen. Together these studies suggest that Foxy-5 may be a potential new supplementary treatment for ERalpha negative breast cancer patients, as it addresses two of the most important aspects of cancer related mortality -- non response to endocrine therapy and metastasis.

Retraction for: "The WNT-5a derived peptide, Foxy-5, possesses dual properties that impair progression of ERa negative breast cancer," by Caroline E. Ford, Elin J. Ekström, Jillian Howlin and Tommy Andersson, which appeared in the June 15, 2009 issue of Cell Cycle (Ford CE, et al. Cell Cycle 2009; 8:1838-1842; 10.4161/cc.8.12.8863). The authors wish to note the following: "Recently a paper, on which I was the senior author and that was published in the Proceedings of the National Academy of Sciences titled "Wnt-5a signaling restores tamoxifen sensitivity in estrogen receptor-negative breast cancer cells" (Ford CE, Ekström EJ, Andersson T. Proc Natl Acad Sci USA 2009; 106:3919-24) was retracted. The fact that this paper was the direct reason for our review article in the Cell Cycle journal makes it logical that I also retract the cited review article published in the Cell Cycle journal, the other authors approve this retraction. We apologize for any inconvenience this may have caused."