Fig 2: Induced WNT11 expression with tumor hypoxia and WNT11 regulates tumor growth.Antiangiogenic therapy induces WNT11 expression in the orthotopic malignant glioma model. Athymic mice implanted with U87ΔEGFR cells were administered either bevacizumab (6 mg/kg) or vehicle three times per week for 4 weeks. (A) Increased Wnt11 mRNA in xenografts from mice treated with bevacizumab. Values were normalized to HPRT mRNA and are expressed relative to control (n = 10 per group). Values are mean ± s.e.m. *p < 0.05, **p < 0.01. (B) Bevacizumab increased expression of HIF-1α and HIF-2α and WNT11. First 10 lanes are control tumors, and the last 10 lanes are tumors from bevacizumab-treated animals. Lysates from whole tissue and nuclei are indicated. α-Tubulin, actin and lamin A/C are loading controls.

Fig 3: HIF-1a is the predominant transcriptional regulator of WNT11 expression during hypoxia.(A,C–E) EMSCs isolated from the indicated mouse genotypes were infected with lentivirus expressing GFP or Cre recombinase. Non-infected cells and GFP infected cells served as controls. Immunoblot analyses of EMSCs derived from the indicated genotypes treated with 0.1 mM DMOG for 24 hrs. (A) Attenuated WNT11 expression in Hif-1a KO EMSCs (lenti-Cre infected Hif-1af/f). (B) HIF-1a regulates WNT11 expression during hypoxia. Impaired WNT11 expression in MDA-MB-231 cells stably expressing HIF-1a shRNAs with hypoxia. Cells were exposed to air (21% O2) or hypoxia (1% O2) for 24 hrs (left panel). Overexpression of HIF-1a in MDA-MB-231 cells enhances WNT11 expression (right panel). (C) Markedly increased WNT11 levels in Hif-1a overexpressing EMSCs (lenti-Cre infected Hif-1a-Tgfl-Stop). (D) Elevated HIF-1a and WNT11 protein after DMOG treatment in HIF-2a KO cells (lenti-Cre infectied Hif-2afl/fl). (E) Little effect of HIF-2a overexpression on WNT11 protein expression in EMSC (lenti-Cre infectied Hif-2a-Tgfl-Stop). (F) HIF-1a binds to WNT11 and VEGF promoters as assessed by ChIP analyses (n = 3 each condition). (G) WNT11 expression was suppressed in cells with a ß-catenin deficiency. EMSCs stably expressing shRNAs against ß-catenin or scrambled control were treated with 0.1 mM DMOG for 24 hrs and analyzed by immunoblotting. (H) Co-transfection with expression vectors for ß-catenin, Hif-1a and ARNT stimulates further induction of Wnt11 promoter activity. Constructs encoding Hif-1a, Hif-2a, ARNT, ß-catenin and Wnt11 promoter-luciferase reporter, were transiently transfected into HEK293T cells. Cells were harvested and luciferase activities were measured 48 hrs after transfection (n = 3). For panels (A–E,G), laminin, a-tubulin, ERK1/2 and lamin A/C were used as loading controls, WNT11 normalized to a-Tubulin was shown under the bots. For panels (F,H), values are mean ± s.e.m. *p < 0.05, **p < 0.01

Fig 4: Induced WNT11 expression with tumor hypoxia and WNT11 regulates tumor growth.(A) WNT11 is elevated in hypoxic regions of tumors. Mammary pads of female BALB/c mice were injected with 4T1 cells. Five weeks after transplantation, tumor tissue was harvested and pimonidazole (Hypoxyprobe; red), HIF-1α (blue), Wnt11 (green) and their colocalization (right) in tumor tissue are shown. The scale bar represents 200 μm. (B) Wnt11 deficiency suppresses the progression of 4T1 breast cancer in mice. 4T1 cells stably expressing shRNAs against Wnt11 or scrambled control were injected to BALB/c mice, and then tumor size was measured (n = 8). Values are mean ± s.e.m. **p < 0.01. (C) Bioluminescence imaging of tumor bearing mice. Representative images taken two weeks after injection of firefly luciferase-tagged 4T1 cells stably expressing shRNAs against Wnt11 or scrambled control into BALB/c mice.

Fig 5: Hypoxia induces expression of WNT11 through VHL.(A,B) Higher basal levels of WNT11 protein in Vhl-deleted cells (lenti-Cre infected Vhlf/f). EMSCs isolated from Vhlf/f mouse were infected with lentivirus carrying either GFP gene (for control) or Cre recombinase (for knockout). Non-infected cells were also used as a control. Immunoblot analysis of control or Vhl KO EMSCs treated with 0.1 mM DMOG (A), and EMSCs exposed to air (21% O2) or hypoxia (1% O2) for 24 hrs (B). Laminin, α-tubulin, and lamin A/C were used as loading controls, WNT11 normalized to α-Tubulin was shown. (C,D) Inactivation of the Vhl gene results in increased Wnt11 mRNA. Wnt11 and Vegf mRNA levels in liver (C) or duodenum (D) were measured by qPCR in Liver-VhlcKO or duodenum-VhlcKO and control mice (n = 5 per group). Values normalized to Tbp mRNA are expressed relative to tissues from control mice. For panels (C,D), values are mean ± s.e.m. *p < 0.05, **p < 0.01.