Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7

Abstract

Expression of an oncogene in a primary cell can, paradoxically, block proliferation by inducing senescence or apoptosis through pathways that remain to be elucidated. Here we perform genome-wide RNA-interference screening to identify 17 genes required for an activated BRAF oncogene (BRAFV600E) to block proliferation of human primary fibroblasts and melanocytes. Surprisingly, we find a secreted protein, IGFBP7, has a central role in BRAFV600E-mediated senescence and apoptosis. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a proapoptotic BCL2 family protein. Recombinant IGFBP7 (rIGFBP7) induces apoptosis in BRAFV600E-positive human melanoma cell lines, and systemically administered rIGFBP7 markedly suppresses growth of BRAFV600E-positive tumors in xenografted mice. Immunohistochemical analysis of human skin, nevi, and melanoma samples implicates loss of IGFBP7 expression as a critical step in melanoma genesis.

Figure 1. A Genome-Wide shRNA Screen Identifies Factors Required for BRAFV600E-Mediated Senescence and Apoptosis

(A) Schematic summary of the genome-wide shRNA screen. (B) Proliferation of the 17 BRAFV600E/PFF KD cell lines. 1×10⁴ PFF fibroblasts stably expressing the indicated shRNA were cultured in 12-well plates, infected with the BRAFV600E-expressing retrovirus and after 14 days stained with crystal violet. (C) Quantitative proliferation assays of the 17 BRAFV600E/melanocyte KD cell lines. Melanocytes stably expressing the indicated shRNA were infected with the BRAFV600E-expressing retrovirus and after 14 days analyzed by trypan blue exclusion test. Growth of BRAFV600E/melanocytes is expressed relative to that of normal melanocytes. Growth of BRAFV600E/melanocyte KD cell lines is normalized to that of the corresponding melanocyte KD cell line in the absence of BRAFV600E expression. Error bars represent standard error. (D) DNA replication assays of the 17 BRAFV600E/melanocyte KD cell lines, monitored by BrdU incorporation. (E) Apoptosis assays of the 17 BRAFV600E/melanocyte KD cell lines, monitored by Annexin V-PE staining. (F) Immunoblot analysis monitoring induction of p16^INK4a and H3K9 acetylation in each of the 17 BRAFV600E/melanocyte KD cell lines. β-ACTIN (ACTB) was monitored as a loading control.

Figure 2. A Secreted Protein, IGFBP7, Induces Senescence and Apoptosis through an Autocrine/Paracrine Pathway

(A) (Top) Immunoblot analysis of IGFBP7 levels in CM from normal melanocytes, BRAFV600E/melanocytes, BRAFV600E/melanocytes stably expressing an IGFBP7 shRNA or in BRAFV600E/melanocyte CM treated with an α-IGFBP7 antibody. (Bottom) Proliferation assays on naïve melanocytes following addition of the different CMs described above. Proliferation was measured and normalized to the growth of untreated melanocytes. Error bars represent standard error. (B) Coomassie-stained gel of purified, recombinant IGFBP7 (rIGFBP7). Molecular weight markers are shown on the left, in kDa. (C) Proliferation assay monitoring the effect of rIGFBP7 on the growth of melanocytes 14 days after treatment. (D) β-galactosidase staining of melanocytes infected with a retrovirus expressing either empty vector or BRAFV600E, or melanocytes treated with CM from BRAFV600E/melanocytes or rIGFBP7. Images are shown at a magnification of 10X, 20X and 40X. (E) Proliferation assay monitoring growth rates of untreated melanocytes, or melanocytes stably expressing an NS or IGFBP7 shRNA.

Figure 3. Selective Sensitivity of Melanoma Cell Lines Containing an Activating BRAF Mutation to IGFBP7-Mediated Apoptosis

(A) (Top) Immunoblot analysis monitoring IGFBP7 levels in the CM from a panel of human melanoma cell lines. (Bottom) Quantitative real-time RT-PCR analysis of IGFBP7 expression. Error bars represent standard error. (B) Proliferation assays of human melanoma cell lines 24 hrs after rIGFBP7 treatment. Proliferation was normalized to the growth of the corresponding cell line in the absence of rIGFBP7 addition. (C) Apoptosis assays of human melanoma cell lines treated with rIGFBP7. (D) (Top), Immunoblot analysis in SK-MEL-28 cells in the presence or absence of rIGFBP7 and stably expressing either an NS, SMARCB1 or BNIP3L shRNA. (Bottom), Schematic summary of the IGFBP7-mediated apoptotic pathway. (E) ChIP analysis monitoring STAT1 recruitment to the SMARCB1 promoter in SK-MEL-28 cells. (F) qRT-PCR analysis of SMARCB1 (left) or STAT1 (right) mRNA levels in SK-MEL-28 cells following treatment with an NS or STAT1 siRNA. (G) ChIP analysis monitoring SMARCB1 (left) or BRG1 (right) recruitment to the BNIP3L promoter in SK-MEL-28 cells. (H) SK-MEL-28 cells were incubated with rIGFBP7 for 0, 2, 6, 12 or 24 hrs, following which the cells were washed and cultured in medium lacking rIGFBP7 and apoptosis was quantitated.

Figure 4. IGFBP7 Blocks BRAF-MEK-ERK Signaling to Activate the Apoptotic Pathway

(A) Immunoblot analysis in SK-MEL-28 cells treated with increasing concentrations of rIGFBP7 (0.2, 1.0, 2.0, 5.0 or 10 μg/ml) for 24 hrs. (B) Proliferation assays monitoring sensitivity of SK-MEL-28 cells to rIGFBP7. Cells were transfected with an empty expression vector or a constitutively activated ERK2 or MEK1 mutant. Cell growth was analyzed 24 hrs after treatment with rIGFBP7 and normalized to the growth of the corresponding cell line in the absence of rIGFBP7 addition. Error bars represent standard error. (C) Immunoblot analysis in SK-MEL-28 cells stably transfected with an empty expression vector or a constitutively activated ERK2 mutant. SK-MEL-28 cells were either untreated or treated with 10 μg/ml of rIGFBP7, as indicated, for 24 hrs prior to harvesting cells. (D) Immunoblot analysis in SK-MEL-28 cells 24 hrs following treatment with rIGFBP, a MEK inhibitor (MEK-i) or a RAF inhibitor (RAF-i).

Figure 5. IGFBP7 Suppresses Growth of BRAFV600E-Positive Tumors in Xenografted Mice

(A) SK-MEL-28 or SK-MEL-31 cells were injected subcutaneously into the flanks of nude mice, and three, six and nine days later (denoted by arrows), the mice were injected at the tumor site with rIGBP7 or, as a control, PBS. Error bars represent standard error. (B) SK-MEL-28 or SK-MEL-31 cells were injected into the flanks of nude mice. When tumors reached a size of 100 mm³, 100 μg rIGFBP7 was systemically administered by tail vein injection at days 6, 9 and 12 (indicated by arrows). (C) Dose-dependent suppression of tumor growth by rIGFBP7. SK-MEL-28 cells were injected into the flanks of nude mice as described in (B), following which rIGFBP7 was systemically administered by tail vein injection. Tumor volume was measured at day 21.

Figure 6. Loss of IGFBP7 Expression is a Critical Step in Development of a BRAFV600E-Positive Melanoma

Immunohistochemical analysis of IGFBP7 expression in human tissue samples. Samples were stained with hematoxylin and eosin (H&E). Arrowheads indicate IGFBP7-positive melanocytes. Images are shown at 2X and/or 20X.

Figure 7. The IGFBP7 Promoter is Hypermethylated in BRAFV600E-Positive Melanoma Cell Lines and Tissues

(A) Bisulfite sequence analysis of the IGFBP7 promoter in human tissue samples. (Top) Schematic of the IGFBP7 promoter; positions of the CpG dinucleotides are shown to scale by vertical lines. (Bottom) Each circle represents a CpG dinucleotide: open (white) circles denote unmethylated CpG sites and filled (black) circles indicate methylated CpG sites. Each row represents a single clone. (B) Bisulfite sequence analysis of the IGFBP7 promoter in a panel of melanoma cell lines. (C) qRT-PCR analysis of IGFBP7 mRNA levels in melanoma cell lines following treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza). Error bars represent standard error. (D) Schematic summary of BRAFV600E-mediated senescence and melanoma progression. Normal melanocytes (BRAF-wt) express and secrete low levels of IGFBP7, which inhibits BRAF-MEK-ERK signaling through an autocrine/paracrine pathway, thereby restraining proliferation. In BRAFV600E-positive nevi, constitutive activation of the BRAF-MEK-ERK pathway increases expression and secretion of IGFBP7, and the resultant high levels of IGFBP7 inhibit BRAF-MEK-ERK signaling and activate senescence. In a BRAFV600E-positive melanoma, IGFBP7 expression is lost, enabling the cells to escape from senescence and resulting in uncontrolled proliferation. Addition of exogenous IGFBP7 to BRAFV600E-positive melanoma cells inhibits BRAF-MEK-ERK signaling and activates apoptosis.