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.2014 Jun 1;74(11):2962-73.
doi: 10.1158/0008-5472.CAN-13-2421. Epub 2014 Mar 17.

Myeloid WNT7b mediates the angiogenic switch and metastasis in breast cancer

Affiliations

Myeloid WNT7b mediates the angiogenic switch and metastasis in breast cancer

Eun-Jin Yeo et al. Cancer Res..

Abstract

Oncogenic targets acting in both tumor cells and tumor stromal cells may offer special therapeutic appeal. Interrogation of the Oncomine database revealed that 52 of 53 human breast carcinomas showed substantial upregulation of WNT family ligand WNT7B. Immunolabeling of human mammary carcinoma showed that WNT7B immunoreactivity was associated with both tumor cells and with tumor-associated macrophages. In the MMTV-PymT mouse model of mammary carcinoma, we found tumor progression relied upon WNT7B produced by myeloid cells in the microenvironment. Wnt7b deletion in myeloid cells reduced the mass and volume of tumors due to a failure in the angiogenic switch. In the tumor overall, there was no change in expression of Wnt/β-catenin pathway target genes, but in vascular endothelial cells (VEC), expression of these genes was reduced, suggesting that VECs respond to Wnt/β-catenin signaling. Mechanistic investigations revealed that failure of the angiogenic switch could be attributed to reduced Vegfa mRNA and protein expression in VECs, a source of VEGFA mRNA in the tumor that was limiting in the absence of myeloid WNT7B. We also noted a dramatic reduction in lung metastasis associated with decreased macrophage-mediated tumor cell invasion. Together, these results illustrated the critical role of myeloid WNT7B in tumor progression, acting at the levels of angiogenesis, invasion, and metastasis. We suggest that therapeutic suppression of WNT7B signaling might be advantageous due to targeting multiple aspects of tumor progression.

©2014 American Association for Cancer Research.

PubMed Disclaimer

Conflict of interest statement

Conflicts of Interest: There are no conflicts of interest.

Figures

Fig 1
Fig 1. WNT7B expression in human mammary tumors
WNT7B expression (as log2 median centered ratio) for mammary gland, normal and tumor stroma, shown either as raw data (A) or as the mean (B). In the chart showing the mean, the dots indicate the extreme data values. Significance as labeled. (C) Meta-analysis of recent gene expression profiling for WNT7B where the colored squares indicate the median rank for WNT7B across each analysis. WNT7B ranks in the top 5–10% in all 6 analyses. (D) End-point RT-PCR for WNT7B in flow-sorted CD45+, CD11b+, CD163+, CD14+ TAMs from human mammary carcinoma (T) and adjacent normal tissue (C). (E–N) Immunoreactivity in cryosections of human mammary carcinoma for WNT7B (red) and the TAM markers CD68 (green) or CD163 (green) as labeled. Images at 600× magnification (E, H, L) show boxed regions (F, G, I–K, M, N) that are digitally magnified in the adjacent panels. Magnified panels are in sets that show all the color channels or just the TAM marker (green) with nuclei (blue) or WNT7B (red) with nuclei (blue). In the magnified panels, a dashed line or a white arrow indicates the green-labeled region identifying a TAM. Adjacent, these same regions are indicated in images where only the WNT7B labeling (red) is shown. In all cases, WNT7B immunoreactivity is associated with TAMs and their processes. The tumor cells are also strongly immunoreactive with the WNT7B antibody. The white scale bars are 10 µm.
Fig. 2
Fig. 2.Wnt7b expression and deletion in the MMTV-PyMT model
(A) End-point RT-PCR for Wnt7b from in flow-sorted dextran+, F4/80+ mouse macrophages fromWnt7btm2Amc/− (control, C) andWnt7btm2Amc/−; Csf1r-icre (mutant, M) MMTV-PyMT tumors. (B) Genotyping PCR on peritoneal cells (PC) or in flow-sorted dextran+, F4/80+ macrophages from MMTV-PyMT tumors of the indicated genotypes. The PCR primers used do not amplify a product from the recombinedWnt7btm2Amc allele. (C) RelativeWnt7b mRNA expression in control (C) and mutant (M)MMTV-PyMT mammary tumors at premalignant (Hyperplasia and Adenoma, H/A), or malignant (early carcinoma, EC, and late carcinoma, LC) stages. Error bars are SEM. (D) Percentage of marker positive cells in control (MMTV-PyMT; Wnt7btm2Amc/−, C) and mutant (MMTV-PyMT; Wnt7btm2Amc/−; Csf1r-iCre, M) tumors combined from the 20–22 week range. Error bars are SEM.
Fig 3
Fig 3. The angiogenic switch is suppressed in the absence of TAM Wnt7b
(A) Percentage of CD31+, CD105+ vascular endothelial cells in control and mutant tumors combined from the 20–22 week range. (B) Representative images of Texas Red dextran perfused blood vessels in PyMT mammary tumors at premalignant (Hyperplasia and Adenoma, H/A), or malignant (early carcinoma, EC, and late carcinoma, LC) stages. The scale bar in (B), H/A, control is 100 µm and applies to all panels. (C, D) Quantification of dextran labeled vessels for a given tumor stage using either vessels per field (C) or branch-points per field (D). (E, F) Quantification of CD31 labeling in control (C) and mutant (M) late carcinomas shown either as CD31+ area (E) or CD31+ vessels (F). For (A and C–F) sample number is shown at base of histogram bar. Error bars are SEM.
Fig 4
Fig 4. The expression of Vegfa is reduced by the deletion of tumor stroma cell Wnt7b
(A) RelativeVegfa mRNA expression in control (C) and mutant (M) MMTV-PyMT tumors at H/A, EC and LC stages. Inset: Immunoblotting for VEGFA and actin control and mutant MMTV-PyMT tumor lysates. (B) RelativesFlt-1 mRNA level in control and mutant MMTV-PyMT tumors at H/A, EC and LC stages. (C, D) Relative expression ofAxin2, c-Myc, andCyclinD1 in control and mutant whole tumor (C) and CD31+, CD105+ blood vascular endothelial cells (D). Sample number is shown at the base of each histogram bar. Error bars are SEM.
Fig. 5
Fig. 5. TAMWnt7b deletion suppresses tumor volume, mass and progression
(A) Control and mutant inguinal gland volume from 6 weeks to 22 weeks. (B, C) Inguinal gland mass at 16 (B) and 22 (C) weeks. For (B, C) sample number is shown at the base of the chart. Error bars are SEM. (D) Ki67 labeled cells per field for control (MMTV-PyMT; Wnt7btm2Amc/−, C) and mutant (MMTV-PyMT; Wnt7btm2Amc/−; Csf1r-iCre, M) early carcinomas (EC) and late carcinomas (LC). P values as labeled, error bars are SEM. (E) Distribution of the stage of progression for control (C) and mutant (M) MMTV-PyMT mammary tumors at 16 weeks (for control, C, n=18, for mutant, M, n=18) and 22 weeks (for C, n=17, for M, n=9). Hyperplasia and adenoma: H/A, early carcinoma: EC, and late carcinoma: LC.
Fig 6
Fig 6. Metastasis is reduced in the absence of tumor stroma cell WNT7b
(A–D) Representative images of metastasized lung in control (A, C) and mutant (B, D) mice in whole mount (A, B) and in section (C, D) at 22 weeks. Scale bars in (A–D) are 180 µm. (A, B) Obvious surface metastases in whole mount lungs are marked with yellow dots. (C, D) In lung sections, metastases appear as dense purple regions. (E) Quantification of total lung weight in control and mutant, tumor free and tumor bearing mice at 22 weeks. (F) Relative PyMT mRNA expression in control and mutant lung at 22 weeks. (G) The lung metastasis index for control and mutant mice at 22 weeks. Sample number is shown at the base of each histogram bar in (E–G). (H) Quantification of the number of cells that migrate into micro-needles placed in either control (MMTV-PyMT; Wnt7btm2Amc/−, C) or mutant (MMTV-PyMT; Wnt7btm2Amc/−; Csf1r-iCre, M) tumors. The micro-needles were loaded with either vehicle (−) or EGF (+) as labeled. These data show that the absence of TAM Wnt7b significantly reduced the number of cells that migrate into the needle under the influence of EGF. Error bars for all charts are SEM.
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