CSN5 isopeptidase activity links COP9 signalosome activation to breast cancer progression

Abstract

CSN5 has been implicated as a candidate oncogene in human breast cancers by genetic linkage with activation of the poor-prognosis, wound response gene expression signature. CSN5 is a subunit of the eight-protein COP9 signalosome, a signaling complex with multiple biochemical activities; the mechanism of CSN5 action in cancer development remains poorly understood. Here, we show that CSN5 isopeptidase activity is essential for breast epithelial transformation and progression. Amplification of CSN5 is required for transformation of primary human breast epithelial cells by defined oncogenes. The transforming effects of CSN5 require CSN subunits for assembly of the full COP9 signalosome and the isopeptidase activity of CSN5, which potentiates the transcriptional activity of MYC. Transgenic inhibition of CSN5 isopeptidase activity blocks breast cancer progression evoked by MYC and RAS in vivo. These results highlight CSN5 isopeptidase activity in breast cancer progression, suggesting it as a therapeutic target in aggressive human breast cancers.

Figure 1. CSN5 and MYC are coordinately amplified in PHMLEB and PHMLER cells

A, schematic of stepwise oncogenic transformation of primary HMEC. PHMLEB cells are immortalized; PHMLER cells are tumorigenic (3). B, array-based CGH of chromosome 8q DNA copy number in normal diploid, PHMLEB, and PHMLER cells. Red, amplification; green, deletion. Location of CSN5 and MYC on 8q is indicated. Data are shown as a moving average of the surrounding 15 genes. C, quantitative microsatellite analysis of CSN5 and MYC DNA copy number relative to normal. Columns, mean; bars, SE.

Figure 2. CSN5 and MYC are required for transformed phenotypes of PHMLER cells

A, immunoblot 3 d after siRNA transfection in the indicated cells. Relative levels of MYC and CSN5 protein were quantified with ImageJ. B, fold change of MYC and CSN5 mRNA in PHMLER cells over primary HMEC (dashed line) as determined by quantitative RT-PCR. Columns, mean; bars, SE. C, normalization of CSN5 and MYC synergistically inhibits wound signature expression. Change in wound signature score (see Materials and Methods) of each sample (left) plotted on a scale of the significance of the change in score (right). Columns, mean; bars, SE. The significance scale reflects the exponential increase in the risk of metastasis and death with linear increase of the wound score (6). D, normalization of CSN5 and MYC synergistically inhibits anchorage-independent growth. Left, quantification of soft agar colonies ≥100 μm in diameter 8 d after plating. Columns, mean; bars, SE. Dashed line, background colony growth of the nontumorigenic PHMLEB cells. *, P < 0.002, Student’s t test, compared with siGFP; **, P < 0.002, Student’s t test, compared with siMYC and siCSN5. Right, images of soft agar colonies. Bar, 100 μm.

Figure 3. Transformed phenotypes of PHMLER cells require additional CSN subunits

A, immunoblot 3 d after siRNA transfection in PHMLER cells. *, nonspecific band. B, cell proliferation by daily counts of transfected PHMLER cells. Points, mean; bars, SE. *, P < 0.001, Student’s t test, compared with siGFP at days 3 and 4. C, silencing of CSN1 and CSN6 inhibits anchorage-independent growth. Top, images of soft agar colonies 7 d after plating. Bar, 200 μm. Bottom, quantification of soft agar colonies 7 or 12 d after plating. Columns, mean; bars, SE. Dashed line, colony growth of PHMLEB cells. *, P < 0.01, Student’s t test, compared with siGFP.

Figure 4. Catalytic activity of CSN5 is required for MYC activation and transformation of PHMLER cells

A, immunoblot of MYC and CSN5 or CSN5^D151N protein in transduced MCF10A cells. For the CSN5 blot, arrow indicates transduced CSN5, which contains a MYC epitope tag. *, nonspecific band. Relative levels of MYC protein are provided. B, CSN5^D151N inhibits activation of MDM2 promoter by MYC. Reporter gene activity in the presence of the indicated gene combinations in transfected 293 cells, normalized to input MYC levels. Columns, mean relative luciferase units (RLU); bars, SE. Mut E-box, MDM2 promoter with mutated E-box element. C, immunoblot of transduced PHMLER cells 3 d after siRNA transfection. UTR, siRNA targeting 3′-UTR of endogenous CSN5. D, catalytic activity of CSN5 is required for anchorage-independent growth. Quantification of soft agar colonies 7 d after plating. Columns, mean; bars, SE. *, P < 10⁻⁴, Student’s t test, compared with siGFP or siCSN5+LZRS-HA-CSN5.

Figure 5. Catalytically inactive CSN5 inhibits breast cancer growth in vivo

A, outline of mouse model of breast cancer. Green letters, genes linked with an internal ribosomal entry site (IRES)-GFP; green circles, cells transduced with IRES-GFP vector. B, top, mammary glands regenerated with GFP-marked MYC alone (10 wk; no tumor formation) or oncogenic RAS + GFP-marked MYC (3 wk; tumor formation). H&E staining of the tumor is shown. Bars, 1 mm (left) and 40 μm (middle and right). Bottom, tumor formation in situ of the indicated transduced genetic elements. MYC and RAS constructs are marked with IRES-CD8; vector and HA-CSN5^D151N constructs are marked with IRES-GFP. White arrows, mammary tumors. C, quantification of GFP staining of tumor sections (only vector and CSN5^D151N are GFP marked). D, increased HA-CSN5^D151N staining correlates with decreased tumor size. Left, quantification of HA staining compared with tumor size of contralateral vector-expressing tumors. The data fit to a linear regression (R² = 0.80; P = 0.01, one-sided t test). Right, RAS+MYC+Vector and the contralateral RAS+MYC+HA-CSN5^D151N–expressing tumor sizes from each animal (tumors from the same animal are connected by a line). In tumors with HA staining >1, on average the tumor size was reduced by 40%.

Figure 6. CSN5^D151N inhibits tumor cell proliferation and lowers tumor grade

A, H&E staining and immunohistochemistry of the indicated proteins in RAS+MYC+Vector, RAS+MYC+HA-CSN5^D151N, and RAS+MYC (CSN5^D151N with no GFP or HA staining) tumors. Bar, 50 μm. Inset of H&E shows single cells magnified, highlighting the difference in nuclear to cytoplasmic ratios. Bar, 10 μm. B, quantification of Ki67-positive cells. Columns, mean; bars, SE. *, P < 10⁻⁶, Student’s t test, compared with all other samples. C, quantification of nuclear to cytoplasmic ratio. Columns, mean; bars, SE. *, P < 10⁻⁸, Student’s t test, compared with all other samples.