PHIP as a therapeutic target for driver-negative subtypes of melanoma, breast, and lung cancer

Abstract

The identification and targeting of key molecular drivers of melanoma and breast and lung cancer have substantially improved their therapy. However, subtypes of each of these three common, lethal solid tumors lack identified molecular drivers, and are thus not amenable to targeted therapies. Here we show that pleckstrin homology domain-interacting protein (PHIP) promotes the progression of these "driver-negative" tumors. Suppression of PHIP expression significantly inhibited both tumor cell proliferation and invasion, coordinately suppressing phosphorylated AKT, cyclin D1, and talin1 expression in all three tumor types. Furthermore, PHIP's targetable bromodomain is functional, as it specifically binds the histone modification H4K91ac. Analysis of TCGA profiling efforts revealed PHIP overexpression in triple-negative and basal-like breast cancer, as well as in the bronchioid subtype of nonsmall cell lung cancer. These results identify a role for PHIP in the progression of melanoma and breast and lung cancer subtypes lacking identified targeted therapies. The use of selective, anti-PHIP bromodomain inhibitors may thus yield a broad-based, molecularly targeted therapy against currently nontargetable tumors.

Keywords: PHIP; chromatin remodeling; driver gene-negative; target.

Fig. 1.

Effects of stable shRNA-mediated suppression of PHIP in MDA-MB-231 cells. (A) Western analysis of expression of PHIP and other proteins in MDA-MB-231 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738). (B) Colony formation ability of MDA-MB-231 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738) (P < 0.01). (C) Invasion into Matrigel of MDA-MB-231 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738) with corresponding microphotographs (P < 0.001). (D) Quantification of total tumor counts on day 36 in the lungs of nude mice i.v. injected with MDA-MD-231 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738) (P < 0.00001). (E and F) Qualitative immunofluorescence analysis of (E) PHIP and Talin 1 and (F) PHIP and Cyclin D1 in MDA-MB-231 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738). (Scale bar, 20 µm.) (G) Effects of overexpression of TLN1 cDNA or control plasmid on invasive capacity into Matrigel of MDA-MB-231 cells expressing anti-PHIP shRNA (127738) (P = 0.02).

Fig. 2.

Effects of modulation of PHIP expression in MDA-MB-231 cells. (A) Western analysis of expression of PHIP and other proteins in MDA-MB-231 cells overexpressing PHIP cDNA or control plasmid. (B) Effects of overexpression of PHIP cDNA or control plasmid on invasive capacity into Matrigel of MDA-MB-231 cells (P < 0.05). (C) Effects of overexpression of PHIP cDNA or control plasmid on colony-formation ability of MDA-MB-231 cells (P < 0.05). (D–G) Qualitative immunofluorescence analysis of (D) PHIP and Talin 1, (E) PHIP and Cyclin D1, (F) PHIP and pHH3, and (G) PCNA and Ki67 in MDA-MB-231 cells overexpressing PHIP cDNA or control plasmid. (Scale bar, 20 µm.)

Fig. 3.

Effects of stable shRNA-mediated suppression of PHIP in H1703 cells. (A) Western analysis of expression of PHIP and other proteins in H1703 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738). (B) Colony formation ability of H1703 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738) (P < 0.02). (C) Invasion into Matrigel of H1703 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738) (P < 0.04). (D) Tumor volume 50 d after s.c. injection of H1703 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738) (P = 0.000006). (E and F) Qualitative immunofluorescence analysis of (E) PHIP and Talin 1 and (F) PHIP and Cyclin D1 in H1703 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738). (Scale bar, 20 µm.) (G) Effects of overexpression of TLN1 cDNA or control plasmid on invasive capacity into Matrigel of H1703 cells expressing anti-PHIP shRNA (127738) (P < 0.001).

Fig. 4.

Effects of modulation of PHIP expression in H1703 cells. (A) Western analysis of expression of PHIP and other proteins in H1703 cells overexpressing PHIP cDNA or control plasmid. (B) Effects of overexpression of PHIP cDNA or control plasmid on invasive capacity into Matrigel of H1703 cells (P < 0.05). (C) Effects of overexpression of PHIP cDNA or control plasmid on colony formation ability of H1703 cells (P < 0.05). (D–G) Qualitative immunofluorescence analysis of (D) PHIP and Talin 1, (E) PHIP and Cyclin D1, (F) PCNA and pHH3, and (G) PHIP and Ki67 in H1703 cells overexpressing PHIP cDNA or control plasmid. (Scale bar, 20 µm.)

Fig. 5.

Effects of shRNA-mediated suppression of PHIP in Ma-Mel-12 cells. (A) Western analysis of expression of PHIP and other proteins in Ma-Mel-12 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738). (B) Colony formation ability of Ma-Mel-12 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738) (P = 0.001). (C) Invasion into Matrigel of Ma-Mel-12 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738) (P = 0.005). (D and E) Qualitative immunofluorescence analysis of (D) PHIP and Talin 1 and (E) PHIP and Cyclin D1 in Ma-Mel-12 cells expressing anti-luc shRNA or anti-PHIP shRNA (127738). (Scale bar, 20 µm.)

Fig. 6.

TCGA analysis of PHIP expression. (A–D) Box plots showing mean expression levels for PHIP in various molecular subtypes of human specimens from TCGA of (A) and (B) breast cancer, (C) lung cancer, and (D) melanoma.

Fig. 7.

Colocalization of PHIP and H4K91ac. (A) Western analysis of PHIP and other proteins in nuclear (NE) versus cytoplasmic (CE) extracts of MDA-MB-231, H1703 and Ma-Mel-12 cells. Lamin B1 was used as a specific nuclear marker. (B) Qualitative immunofluorescence analysis showing colocalization of PHIP and H4K91ac (see arrows) in MDA-MB-231, H1703, and Ma-Mel-12 cells with or without insulin stimulation. (Scale bar, 20 µm.)

Fig. 8.

Colocalization and physical interaction of PHIP and H4K91ac. (A) Z-stacks of confocal images of PHIP and H4K91ac colocalization (see arrows) in MDA-MB-231, H1703, and Ma-Mel-12 cells with or without insulin stimulation. (Scale bar, 5 µm.) (B) Nuclear extracts from H1703 cells were subjected to the immunoprecipitation of endogenous H4K91ac using an anti-H4K91ac antibody or IgG control antibody followed by Western analysis of endogenous PHIP, using an anti-PHIP antibody (Left); nuclear extracts from H1703 cells were subjected to the immunoprecipitation of endogenous PHIP, using an anti-PHIP antibody or IgG control antibody followed by Western analysis of endogenous H4K91ac, using an anti-H4K91ac antibody (Right).