p16INK4A represses breast stromal fibroblasts migration/invasion and their VEGF-A-dependent promotion of angiogenesis through Akt inhibition - PubMed (original) (raw)

p16INK4A represses breast stromal fibroblasts migration/invasion and their VEGF-A-dependent promotion of angiogenesis through Akt inhibition

Mysoon M Al-Ansari et al. Neoplasia. 2012 Dec.

Abstract

Stromal fibroblasts, the most abundant and probably the most active cellular component of breast cancer-associated stroma, become active and promote angiogenesis through paracrine effects. However, it still unclear how these processes are regulated. Here, we have shown that down-regulation of the tumor suppressor p16(INK4A) protein enhances the migration/invasion abilities of breast stromal fibroblasts, which form dendritic network of extensions into matrigel. Furthermore, we present clear evidence that p16(INK4A) represses the expression/secretion of the proangiogenesis protein vascular endothelial growth factor A (VEGF-A). Consequently, p16(INK4A)-deficient breast stromal fibroblasts and mouse embryonic fibroblasts enhanced endothelial cell differentiation into capillary-like structures in a paracrine manner. This effect was suppressed by adding bevacizumab, a specific VEGF-A inhibitor. Additionally, p16(INK4A)-defective mouse embryonic fibroblasts enhanced angiogenesis in breast cancer xenografts in mice. Furthermore, we have shown that p16(INK4A) suppresses the Akt/mammalian target of rapamycin (mTOR) signaling pathway and its downstream effector hypoxia-inducible factor 1-alpha (HIF-1α), which transactivates VEGF-A. Consequently, Akt inactivation suppressed both the p16(INK4A)-dependent autocrine effect on fibroblast migration/invasion and the paracrine effect on angiogenesis, showing the important role of this protein kinase in mediating the various effects related to p16(INK4A) deficiency. These results indicate that p16(INK4A) is an efficient inhibitor of the migration/invasion abilities of breast stromal fibroblasts and also their paracrine proangiogenic effects, through inhibition of Akt. Therefore, pharmacologic restoration of p16(INK4A) level in stromal fibroblasts may be exploited as therapeutic strategy to help eradicate tumor cells and/or prevent their recurrence, through suppressing cell non-autonomous procarcinogenic mediators.

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Figures

Figure 1

Figure 1

p16 down-regulation enhances fibroblast migration and invasion. (A) Whole-cell lysates were prepared from TCF-64, CAF-64, T64si, and T64C cells and were used for immunoblot analysis. The numbers below the bands indicate fold of induction after normalization against GAPDH. (B, C) T64si and T64C cells (4 x 105) were cultured on the upper compartments of BioCoat Matrigel chambers in the presence of SFM. After 24 hours of incubation, cells were stained with Diff-Quick stain and then counted. (B) Representative photographs showing invasive and migrated cells. Scale bars represent 20 _µ_m. (C) Histograms depict average numbers of invasive and migrated cells, and error bars represent means ± SD. (D) Cells (6 x 104) were seeded with CpM in six-well plates coated with BD Matrigel basement membrane matrix for 24 hours. Representative photographs show the morphology of the invading cells. Scale bars represent 30 _µ_m.

Figure 2

Figure 2

p16 suppresses the expression of VEGF-A. (A) Cell lysates were prepared from the indicated cells and used for immunoblot analysis using the indicated antibodies. (B, C) Total RNA was extracted from the indicated cells and the amount of the VEGF-A mRNA was assessed by real-time RT-PCR. Error bars represent means ± SD. *P < .001.

Figure 3

Figure 3

p16 represses VEGF-A secretion from breast stromal fibroblasts. (A, B) Conditioned media from the indicated cells were collected after 24 hours and the level of secreted VEGF-A was determined by ELISA. Error bars represent means ± SD. *P < .001.

Figure 4

Figure 4

SFCM from p16-deficient human and mice fibroblasts activate HUVEC tube formation in a VEGF-A-dependent manner. SFCM were collected after 24 hours of incubation from the indicated cells and were added independently on HUVECs plated on matrigel (96-well plate), and the differentiation into capillary-like structures was assessed after 4 hours of incubation. (A) Representative photographs of HUVEC cavities. Scale bars represent 30 _µ_m. Stars show cavities and the circle indicates non-sprouting cells. (B) Histograms show number of cavities and non-sprouting cells. (C) SFCM were collected from MEF cells. (D) SFCM from T64si cells were treated with IgG1 or bevacizumab. The numbers between brackets indicate bevacizumab concentrations in _µ_M. (E) Breast cancer xenografts were created by co-injecting MDA-MB-231 cells with p16-/- or p16+/+ MEFs subcutaneously into nude mice. Immunohistochemistry was carried out on tumors containing MEFs either p16-/- or p16+/+ using anti-CD-31 antibody. Scale bars represent 30 _µ_m. Histogram shows average number of microvessels observed in five different fields from two different tumors. Error bars represent means ± SD. *P < .001.

Figure 5

Figure 5

p16 represses the Akt/mTOR/HIF-1α pathway. (A, B) Whole cell lysates were prepared from the indicated cells and were used for immunoblot analysis using the indicated antibodies.

Figure 6

Figure 6

p16 represses stromal fibroblasts migration/invasion and their proangiogenic effect through Akt inhibition. (A) T64si cells were treated with Akt inhibitor III (10 µ_M) and DMSO (-) for 18 hours (upper panel) or cells were treated with control-siRNA and Akt-siRNA (lower panel), and whole-cell lysates were prepared and used for immunoblot analysis using the indicated antibodies. (B, D, E) T64si cells were treated either with Akt inhibitor III (10 µ_M) or DMSO. (B, D) The migration and invasion abilities were assessed using matrigel chambers as in Figure 1_C. Error bars represent means ± SD. *P < .001 (B), or BD matrigel basement membrane matrix as in Figure 1_D (D). (C) T64si cells were treated either with control-siRNA or Akt-siRNA, and the migration/invasion abilities of these cells were assessed as in Figure 1_C_. Representative photographs of migrating and invading cells are shown. Error bars represent means ± SD. (E) SFCM were collected and were added independently on HUVECs plated on matrigel (96-well plate), and the differentiation into capillary-like structures was assessed after 4 hours of incubation. Representative photographs of HUVEC cavities are shown. Scale bars represent 30 _µ_m.

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