HIF1α and HIF2α independently activate SRC to promote melanoma metastases - PubMed (original) (raw)

. 2013 May;123(5):2078-93.

doi: 10.1172/JCI66715. Epub 2013 Apr 8.

Bhavani Krishnan, Sean T Bailey, Stergios J Moschos, Pei-Fen Kuan, Takeshi Shimamura, Lukas D Osborne, Marni B Siegel, Lyn M Duncan, E Tim O'Brien 3rd, Richard Superfine, C Ryan Miller, M Celeste Simon, Kwok-Kin Wong, William Y Kim

Affiliations

HIF1α and HIF2α independently activate SRC to promote melanoma metastases

Sara C Hanna et al. J Clin Invest. 2013 May.

Abstract

Malignant melanoma is characterized by a propensity for early lymphatic and hematogenous spread. The hypoxia-inducible factor (HIF) family of transcription factors is upregulated in melanoma by key oncogenic drivers. HIFs promote the activation of genes involved in cancer initiation, progression, and metastases. Hypoxia has been shown to enhance the invasiveness and metastatic potential of tumor cells by regulating the genes involved in the breakdown of the ECM as well as genes that control motility and adhesion of tumor cells. Using a Pten-deficient, Braf-mutant genetically engineered mouse model of melanoma, we demonstrated that inactivation of HIF1α or HIF2α abrogates metastasis without affecting primary tumor formation. HIF1α and HIF2α drive melanoma invasion and invadopodia formation through PDGFRα and focal adhesion kinase-mediated (FAK-mediated) activation of SRC and by coordinating ECM degradation via MT1-MMP and MMP2 expression. These results establish the importance of HIFs in melanoma progression and demonstrate that HIF1α and HIF2α activate independent transcriptional programs that promote metastasis by coordinately regulating cell invasion and ECM remodeling.

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Figures

Figure 1

Figure 1. Inactivation of Hif1α or Hif2α does not affect initiation of the survival or growth of Pten;Braf melanomas, but abrogates lymph node metastases.

(A) Representative gross images as well as low- and high-power photomicrographs of H&E-stained Pten;Braf, Pten;Braf;Hif1, and Pten;Braf;Hif2 melanomas taken at gross, ×10, and ×20 magnification. (B) Kaplan-Meier survival curve of cohorts of mice of the indicated genotypes (Pten;Braf, n = 39; Pten;Braf;Hif1, n = 20; and Pten;Braf;Hif2, n = 28). P = 0.6585, log-rank test. (C and D) TMA analysis of HIF1α and HIF2α expression in melanocytic lesions. (E and F) Representative gross images, low- and high-power photomicrographs, and quantification of lymph node metastases from the indicated genotypes Pten;Braf (27/39), Pten;Braf;Hif1 (2/13), and Pten;Braf;Hif2 (4/15) taken at gross, ×10, and ×20 magnification. Error bars show SEM. ***P < 0.0005; **P < 0.005; *P < 0.05.

Figure 2

Figure 2. Hypoxia enhances melanoma cell invasion and invadopodia formation.

(A) The indicated cell lines were cultured in normoxia (N) (21% O2) or hypoxia (Hy) (1% O2) overnight. Whole-cell extracts were immunoblotted with the indicated antibodies. (B) Representative photomicrographs of A375 SM and WM2664 cells that have invaded through Matrigel chambers. Original magnification, ×10. (C) Quantification of A375 SM and WM2664 cells that have invaded through Matrigel chambers. (D) Representative immunofluorescence images of A375 SM cells plated on Alexa Fluor 568–conjugated fibronectin and stained with the indicated antibodies. Invadopodia were defined as colocalization of cortactin, F-actin (phalloidin), and degradation of Alexa Fluor 568 fibronectin and are indicated with yellow arrowheads. Original magnification, ×63. (E) Quantification of the percentage of cells with active invadopodia in A375 SM and WM2664 cells cultured overnight in normoxia or hypoxia. (F) Quantification of the number of invadopodia per cell in A375 SM and WM2664 cells cultured overnight in normoxia or hypoxia. Error bars show SEM. ****P < 0.0001; ***P < 0.0005; *P < 0.05.

Figure 3

Figure 3. Knockdown of HIF1α or HIF2α reduces the hypoxia-induced invasion and invadopodia formation of melanoma cell lines.

(A) A375 SM and WM2664 cells were transfected with siRNAs against HIF1α, HIF2α, or a nonspecific sequence (NS). Whole-cell lysates were immunoblotted with the indicated antibodies. (B) Representative photomicrographs of A375 SM and WM2664 cells transfected with the indicated siRNAs, which have invaded through Matrigel chambers under hypoxia. Original magnification, ×10. (C) Quantification of A375 SM and WM2664 cells transfected with the indicated siRNAs, which have invaded through Matrigel chambers under hypoxia. (D) Representative immunofluorescence images of siRNA-transfected A375 SM cells plated on Alexa Fluor 568–conjugated fibronectin and stained with the indicated antibodies. Original magnification, ×63. Invadopodia were defined as colocalization of cortactin, F-actin (phalloidin), and degradation of Alexa Fluor 568 fibronectin and are indicated with yellow arrowheads. (E) Quantification of the percentage of cells with active invadopodia in siRNA-transfected A375 SM and WM2664 cells. (F) Quantification of the number of visible lung metastases found in mice tail vein injected with A375 SM cells. (G) Gross representative images of the lungs from tail-vein–injected mice. Yellow arrows indicate lung metastases. Error bars show SEM. ***P < 0.0005; **P < 0.005; *P < 0.05.

Figure 4

Figure 4. HIF1α and HIF2α upregulate PDGFRα and FAK and are necessary for hypoxia-induced SFK activation.

(A) A375 SM and WM2664 cells were cultured in normoxia (21% O2) or hypoxia (1% O2) overnight. Whole-cell extracts were immunoblotted with the indicated antibodies. (B) A375 SM and WM2664 cells were transfected with siRNAs against HIF1α, HIF2α, or a nonspecific sequence. Whole-cell lysates were immunoblotted with the indicated antibodies. (C) Tumor lysates from the indicated cohorts were immunoblotted for the indicated antibodies. (D and E) A375 SM and WM2664 cells were transfected with siRNAs against HIF1α, HIF2α, or a nonspecific sequence and cultured under hypoxia overnight. Total RNA was used to perform TaqMan quantitative real-time PCR for PDGFRA and FAK. (F) A375 SM and WM2664 cells were transfected with siRNAs against HIF1α, HIF2α, both HIF1α and HIF2α, or a nonspecific sequence. Whole-cell lysates were immunoblotted with the indicated antibodies. (G) Representative photomicrographs of A375 SM and WM2664 cells transfected with the indicated siRNAs, which have invaded through Matrigel chambers. Original magnification, ×10. (H and I) Quantification of A375 SM and WM2664 cells transfected with the indicated siRNAs, which have invaded through Matrigel chambers. Error bars show SEM. ***P < 0.0005; **P < 0.005; *P < 0.05.

Figure 5

Figure 5. Expression of stabilized HIF1α and HIF2α are sufficient to enhance normoxic melanoma cell invasion.

(A) A375 SM and WM2664 cells were infected with retrovirus to stably express EGFP or stabilized versions of HIF1α (HIF1dPA) or HIF2α (HIF2dPA). Whole-cell extracts were immunoblotted with the indicated antibodies. (B) Representative photomicrographs of A375 SM and WM2664 cells stably expressing EGFP, HIF1dPA, or HIF2dPA, which have invaded through Matrigel chambers. Original magnification, ×10. (C) Quantification of A375 SM and WM2664 cells stably expressing EGFP, HIF1dPA, or HIF2dPA, which have invaded through Matrigel chambers. (D) Representative immunofluorescence images of A375 SM cells stably expressing EGFP, HIF1dPA, or HIF2dPA and plated on Alexa Fluor 568–conjugated fibronectin and stained with the indicated antibodies. Original magnification, ×63. Invadopodia were defined as colocalization of cortactin, F-actin (phalloidin), and degradation of Alexa Fluor 568 fibronectin and are indicated with yellow arrowheads. (E) Quantification of the percentage of cells with active invadopodia in A375 SM and WM2664 cells stably expressing EGFP, HIF1dPA, or HIF2dPA. (F) Whole-cell extracts from A375 SM and WM2664 cells stably expressing EGFP, HIF1dPA, or HIF2dPA were immunoblotted with the indicated antibodies. (G) A375 SM cells stably expressing EGFP, HIF1dPA, or HIF2dPA were allowed to adhere to fibronectin-conjugated magnetic beads and subjected to repeated magnetic pulses; the resultant motion and recovery were quantified and used to calculate cell stiffness in Pascals (Pa). Error bars show SEM. ***P < 0.0005; **P < 0.005; *P < 0.05.

Figure 6

Figure 6. HIF1α and HIF2α-dependent invasion require PDGFRα and FAK, respectively.

(A) A375 SM and WM2664 cells stably expressing EGFP, HIF1dPA, or HIF2dPA were transfected with siRNAs against PDGFRα and FAK. Whole-cell extracts were immunoblotted with the indicated antibodies. (B) Representative photomicrographs of A375 SM and WM2664 cells stably expressing EGFP, HIF1dPA, or HIF2dPA and transfected with indicated siRNA oligos, which have invaded through Matrigel chambers. Original magnification, ×10 magnification. (C) Quantification of A375 SM cells stably expressing EGFP, HIF1dPA, or HIF2dPA and transfected with siRNA oligos against either PDGFRα or FAK, which have invaded through Matrigel chambers. (D) Quantification of WM2664 cells stably expressing EGFP, HIF1dPA, or HIF2dPA and transfected with siRNA oligos against either PDGFRα or FAK, which have invaded through Matrigel chambers. Error bars show SEM. ***P < 0.0005; **P < 0.005.

Figure 7

Figure 7. HIF1α and HIF2α regulate expression of MMPs implicated in invadopodia formation.

(A) A375 SM and WM2664 cells were transfected with siRNAs against HIF1α, HIF2α, or a nonspecific sequence. Whole-cell lysates were immunoblotted with the indicated antibodies. (B) A375 SM and WM2664 cells were infected with retrovirus to stably express EGFP or stabilized versions of HIF1α (HIF1dPA) or HIF2α (HIF2dPA). Whole-cell extracts were immunoblotted with the indicated antibodies. (C and D) A375 SM and WM2664 cells were transfected with siRNAs against HIF1α, HIF2α, or a nonspecific sequence. Total RNA was used to perform TaqMan quantitative real-time PCR for MT1-MMP and MMP2. (E) A375 SM cells stably expressing HIF1dPA and HIF2dPA were transfected with siRNAs against MT1-MMP, MMP2, or a nonspecific sequences. Whole-cell lysate was immunoblotted with the indicated antibodies. (F) Representative photomicrographs of A375 SM and WM2664 cells stably expressing EGFP, HIF1dPA, or HIF2dPA and transfected with siRNA oligos against either MT1-MMP or MMP2, which have invaded through Matrigel chambers. Original magnification, ×10. (G and H) Quantification of A375 SM and WM2664 cells stably expressing EGFP, HIF1dPA, or HIF2dPA and transfected with siRNA oligos against MT1-MMP or MMP2, which have invaded through Matrigel chambers. Error bars show SEM. ***P < 0.0005; **P < 0.005; *P < 0.05.

Figure 8

Figure 8. Knockdown of Hif1α and Hif2α reduces the hypoxia-induced invasion of cell lines derived from Pten;Braf melanomas.

(A) 2460 Pten;Braf melanoma cells were treated with the indicated doses of vemurafenib, PIK-90, or DMSO overnight. Whole-cell lysates were immunoblotted with the indicated antibodies. (B) Representative photomicrographs of 2130 and 2460 Pten;Braf melanoma cells, which have invaded through Matrigel chambers under hypoxia or normoxia. Original magnification, ×10. (C) Quantification of 2130 and 2460 Pten;Braf melanoma cells, which have invaded through Matrigel chambers. (D) 2130 and 2460 Pten;Braf melanoma cells were transfected with siRNAs against murine _Hif1_α or _Hif2_α and immunoblotted with the indicated antibodies. (E) Representative photomicrographs of 2130 and 2460 Pten;Braf melanoma cells that were transfected with siRNAs against murine _Hif1_α or _Hif2_α and allowed to invade through Matrigel chambers under hypoxia. Original magnification, ×10. (F) Quantification of 2130 and 2460 Pten;Braf melanoma cells that were transfected with siRNAs against murine _Hif1_α or _Hif2_α and allowed to invade through Matrigel chambers under hypoxia. Error bars show SEM. ***P < 0.0005;**P < 0.005; *P < 0.05.

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