SRC-2-mediated coactivation of anti-tumorigenic target genes suppresses MYC-induced liver cancer

<div><p>Hepatocellular carcinoma (HCC) is the fifth most common solid tumor in the world and the third leading cause of cancer-associated deaths. A <i>Sleeping Beauty</i>-mediated transposon mutagenesis screen previously identified mutations that cooperate with MYC to accelerate liver tumorigenesis. This revealed a tumor suppressor role for <i>Steroid Receptor Coactivator 2</i>/<i>Nuclear Receptor Coactivator 2</i> (<i>Src-2</i>/<i>Ncoa2</i>) in liver cancer. In contrast, SRC-2 promotes survival and metastasis in prostate cancer cells, suggesting a tissue-specific and context-dependent role for SRC-2 in tumorigenesis. To determine if genetic loss of SRC-2 is sufficient to accelerate MYC-mediated liver tumorigenesis, we bred <i>Src-2</i>^<i>-/-</i> mice with a MYC-induced liver tumor model and observed a significant increase in liver tumor burden. RNA sequencing of liver tumors and <i>in vivo</i> chromatin immunoprecipitation assays revealed a set of direct target genes that are bound by SRC-2 and exhibit downregulated expression in <i>Src-2</i>^<i>-/-</i> liver tumors. We demonstrate that activation of <i>SHP (Small Heterodimer Partner)</i>, <i>DKK4</i> (<i>Dickkopf-4)</i>, and <i>CADM4 (Cell Adhesion Molecule 4)</i> by SRC-2 suppresses tumorigenesis <i>in vitro</i> and <i>in vivo</i>. These studies suggest that SRC-2 may exhibit oncogenic or tumor suppressor activity depending on the target genes and nuclear receptors that are expressed in distinct tissues and illuminate the mechanisms of tumor suppression by SRC-2 in liver.</p></div

CONSORT diagram.

<p>CONSORT diagram.</p

Overexpression of SRC-2 upregulates candidate gene expression and reduces HCC cell tumor formation <i>in vivo</i>.

<p>(A) Western blot demonstrating expression of SRC-2 and DKK4 levels in Huh7 cells. Cells were infected with pLJM1 lentiviruses expressing eGFP (as a control) or SRC-2. (B) Real-time PCR quantification of <i>SRC-2</i>, <i>SHP</i> and <i>DKK4</i> expression in Huh7 cells expressing eGFP or SRC-2. (C) MTS assay measuring proliferation of cells overexpressing SRC-2. Error bars in (B) and (C) represent SDs from triplicate measurements. Student’s unpaired t-test was used to evaluate statistical significance. * = p<0.05; ** = p<0.01; **** = p<0.0001. (D) Quantification of tumor volumes of nude mice injected subcutaneously with Huh7 cells overexpressing SRC-2 or control eGFP. Bar graphs represent mean tumor volumes. Error bars represent SDs from a total of ten subcutaneous injections (n = 5 mice) per experimental group tested. Student’s unpaired t-test was used to evaluate statistical significance * = p<0.05; ** = p<0.01; *** = p<0.001.</p

Patient characteristics.

<p>Patient characteristics.</p

Estimated local recurrence free survival for cT1 and cT2 lesions.

<p>Estimated local recurrence free survival for cT1 and cT2 lesions.</p

Tumor stromal hematopoietic cells stimulate LL2 tumor growth.

<p>(A) Co-implanted HSCs or total BM cells stimulate the growth of primary LL2 tumor. 100,000 GFP-marked LL2 cells were co-implanted with with 1×10⁴ CD45.1 total BM cells or enriched normal bone marrow HSCs (as Lin⁻Sca-1⁺Kit⁺ cells) subcutaneously into C57BL/6 CD45.2 host mice (n = 8). During the 3-week period of analysis, the size of the primary tumor was measured. (B) Tumor stromal hematopoietic cells stimulate tumor growth. 100,000 tumor stromal CD45⁺ cells or PB cells from tumor-bearing mice were collected by FACS and co-implant with 10⁵ LL2 cells into healthy mice (n = 5). Shown are the sizes of the primary tumors. Tumor growth curves for different experimental groups were compared using the Generalized Estimating Equations (GEE) method with AR(1) correlation structure. * significantly different from LL2-GFP growth curve, ** significantly different from LL2-GFP and LL2/PB CD45⁺ growth curves, p<0.05.</p

SHP and CADM4 rescue enhanced tumor burden upon SRC-2 inhibition.

<p>(A) Real-time PCR quantification of <i>SRC-2</i>, <i>SHP</i>, <i>CADM4</i>, and <i>DKK4</i> expression in HepG2 cells after inhibition of <i>SRC-2</i> with two independent shRNAs. Bar graphs represent mRNA expression of the labeled transcript normalized to <i>ACTIN</i> and error bars represent SDs from triplicate measurements. (B) Western blot analysis of SRC-2 and its targets CADM4 and DKK4 in HepG2 cells after SRC-2 inhibition with two independent shRNAs. Numbers in red represent quantification of protein levels relative to the control shRNA sample. (C) MTS assay measuring proliferation of HepG2 cells with control shRNA, <i>SRC-2</i> shRNA-1, or <i>SRC-2</i> shRNA-1 with overexpression of <i>SHP</i> or <i>CADM4</i> alone, or in combination with <i>THRSP</i> and <i>DKK4</i> (labeled as ALL 4). (D) Quantification of tumor volumes in nude mice injected with HepG2 cells as described in (C). Bars represent mean tumor volumes. Error bars in real-time quantitation and proliferation assays represent SDs from triplicate measurements. Error bars in xenograft experiments represent SDs from a total of ten subcutaneous injections (n = 5 mice) per shRNA tested. A student’s t-test was performed to determine statistical significance. * = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001. Black asterisks represent comparisons to the control shRNA. Red asterisks represent comparisons to SRC-2 shRNA-1.</p

Inhibition of <i>SHP</i>, <i>DKK4</i>, and <i>CADM4</i> accelerate HCC cell proliferation <i>in vitro</i> and tumor growth <i>in vivo</i>.

<p>(A) Real-time PCR quantification of <i>SHP</i> expression in Huh7 cells after inhibition with two independent shRNAs. Bars graphs represent mRNA expression of <i>SHP</i> normalized to <i>ACTIN</i> and error bars represent SDs from triplicate measurements. (B) MTS proliferation assay measuring proliferation of <i>SHP</i> shRNA and control shRNA cells over time. (C) Quantification of tumor volumes in nude mice injected with Huh7 cells with <i>SHP</i> shRNAs or control shRNA. (D) Top, real-time PCR quantification of <i>DKK4</i> expression in HepG2 cells after inhibition with two independent shRNAs. Bar graphs represent <i>DKK4</i> mRNA expression normalized to <i>ACTIN</i> and error bars represent SDs from triplicate measurements. Bottom, western blot with quantification of DKK4 protein levels and normalized to Tubulin. (E) MTS proliferation assay measuring the proliferation of <i>DKK4</i> shRNA and control shRNA cells with over time. (F) Quantification of tumor volumes in nude mice injected with HepG2 cells with <i>DKK4</i> shRNAs or control shRNA. (G) Real-time PCR quantification and western blot analysis of <i>CADM4</i> mRNA and protein in HepG2 cells after inhibition with two independent shRNAs. (H) MTS assay measuring proliferation of <i>CADM4</i> shRNA and control cells over time. (I) Quantification of tumor volumes in nude mice injected with HepG2 cells with <i>CADM4</i> shRNAs or control shRNA. Bar graphs (C), (F), and (I) represent mean tumor volumes. For this and all subsequent xenograft experiments, the numbers below each bar represent the time (in days) after subcutaneous injection of cells into nude mice. Error bars in real-time quantitation and proliferation assays represent SDs from triplicate measurements. Error bars in xenograft experiments represent SDs from a total of ten subcutaneous injections (n = 5 mice) per shRNA tested. A student’s t-test was performed to determine statistical significance. * = p<0.05; ** = p<0.01; *** = p<0.001; **** = p<0.0001.</p

Estimated local recurrence free survival by fractionation cohort.

<p>Estimated local recurrence free survival by fractionation cohort.</p

Repopulating hematopoietic stem cells and differentiated hematopoietic cells exist in LL2 tumor stroma.

<p>Data from tumor mice were obtained from dissociated tumor masses arising after the subcutaneous injection of 10⁶ LL2 cells into C57BL/6 CD45.1 host mice at day 19 post-implantation (n = 5).</p