SPOCK1 Is a Novel Transforming Growth Factor-β–Induced Myoepithelial Marker That Enhances Invasion and Correlates with Poor Prognosis in Breast Cancer

<div><p>In addition to contraction, myoepithelia have diverse paracrine effects, including a tumor suppression effect. However, certain myoepithelial markers have been shown to contribute to tumor progression. Transforming growth factor-β (TGF-β) is involved in the transdifferentiation of fibroblasts to contractile myofibroblasts. We investigated whether TGF-β can upregulate potential myoepithelial markers, which may have functional and clinicopathological significance in breast cancer. We found that TGF-β induced SPOCK1 expression in MCF10A, MCF12A, and M10 breast cells and demonstrated SPOCK1 as a novel myoepithelial marker that was immunolocalized within or beneath myoepithelia lining ductolobular units. A functional study showed that overexpression of SPOCK1 enhanced invasiveness in mammary immortalized and cancer cells. To further determine the biological significance of SPOCK1 in breast cancer, we investigated the expression of SPOCK1 in 478 invasive ductal carcinoma (IDC) cases through immunohistochemistry and correlated the expression with clinicopathological characteristics. SPOCK1 expression was significantly correlated with high pathological tumor size (P = 0.012), high histological grade (P = 0.013), the triple-negative phenotype (P = 0.022), and the basal-like phenotype (P = 0.026) and was correlated with a significantly poorer overall survival on univariate analysis (P = 0.001, log-rank test). Multivariate Cox regression analysis demonstrated that SPOCK1 expression maintained an independent poor prognostic factor of overall survival. Analysis of SPOCK1 expression on various non-IDC carcinoma subtypes showed an enrichment of SPOCK1 expression in metaplastic carcinoma, which is pathogenetically closely related to epithelial-mesenchymal transition (EMT). In conclusion, we identified SPOCK1 as a novel TGF-β–induced myoepithelial marker and further demonstrated that SPOCK1 enhanced invasion in breast cancer cells and correlated with poor prognosis in breast cancer clinical samples. The enrichment of SPOCK1 expression in metaplastic carcinoma and the correlation between SPOCK1 expression and high histological grading and basal-like phenotypes in IDC evidence an association between SPOCK1 and EMT.</p></div

SPOCK1 protein expression in non-IDC breast carcinoma subtypes.

<p>SPOCK1 protein expression in non-IDC breast carcinoma subtypes.</p

SPOCK1 was a TGF-β-induced myoepithelial marker and SPOCK1 overexpression enhanced invasiveness.

<p>(A) Immunohistochemistry demonstrated that SPOCK1 was the only myoepithelial marker among the evaluated TGF-β–upregulated gene products in MCF10A cells. CXCR7 staining was observed in luminal epithelia but not myoepithelia whereas FBN1 staining was observed diffusely in the stroma but not in the epithelia. IGFL2, PRRX2, PLAT, PCDH9, POSTN and NOV staining was not found in the ductolobular units (left and middle panels). By contrast, SPOCK1 was immunolocalized within (arrow) or beneath (arrowhead) the myoepithelia (right panel). (Magnification × 400). (B) Upregulation of SPOCK1 at mRNA levels (upper panels) and protein levels (lower panels) were observed in MCF10A, M10, and MCF12A cells, 3 days after treatment with TGF-β. S26 was used as an mRNA loading control and GAPDH was used as a protein loading control. (C) Western blotting was used to detect the expression of SPOCK1 in MCF10A, T47D, and MB231 cells 24 h after transient transfection with pCDH-SPOCK1 or control vector pCDH. (D) The invasive capability and proliferation were measured in the cells shown in (C). Data from invasion assay are shown as the mean ± SD of 3 fields. Data from MTT assay are shown as mean ± SD of 3 independent experiments. These results are presented as the percentage relative to their control cells (*, P < 0.05; N.S., nonsignificant).</p

Cox regression model analysis of the clinicopathological variables regarding overall survival in IDC patients.

<p>Cox regression model analysis of the clinicopathological variables regarding overall survival in IDC patients.</p

Immunohistochemical staining for SPOCK1 in various subtypes of breast carcinoma.

<p>SPOCK1 was not expressed in representative cases of invasive lobular carcinoma (A), mucinous carcinoma (B), invasive papillary carcinoma, (C) and invasive micropapillary carcinoma (D). SPOCK1 expression in vascular smooth muscle wall (arrow) served as internal positive control. Two representative SPOCK1-positive cases of metaplastic carcinoma are shown (E and F). Intense SPOCK1 expression was observed in both carcinomatous (Ca) and sarcomatous (Sa) elements in a representative biphasic metaplastic carcinoma (E). SPOCK1 was strongly stained in one representative monophasic epithelial metaplastic carcinoma (squamous metaplasia) (F). (Magnification ×200).</p

Correlation of CTHRC1 expression and clinicopathological factors.

<p>Correlation of CTHRC1 expression and clinicopathological factors.</p

CTHRC1 promotes cell adhesion through activation of integrin β1/FAK pathway.

<p>(A) Adhesion assay. Approximately 1×10⁴ cells were seeded into fibronectin-coated 96-well plates and incubated at 37°C for 30 min, washed, fixed, and stained with crystal violet. The remaining cells were counted. Knockdown of CTHRC1 markedly reduced the number of cells adherent to fibronectin. (B) Treating Huh7 cells with CTHRC1 (100 ng/ml) enhanced adhesion to fibronectin at 15 min and 30 min. (C) Knockdown of CTHRC1 decreased the formation of focal adhesion structures. Cells were trypsinized and replated onto fibronectin-coated slides and incubated at 37°C for 60 min, fixed, and stained. Mouse anti-vinculin was used to visualize focal adhesions (upper panel), and rhodamine-phalloidin was used to visualize actin filaments (lower panel). (D) CTHRC1 treatment enhanced phosphorylation of FAK at residues 576/577. (E) Knockdown of CTHRC1 inhibited phosphorylation of FAK. (F) CTHRC1 enhanced expression of integrin β1. (G) The adhesion-promoting effect of CTHRC1 was abolished by integrin β1 blocking antibody.</p

The experimental setup for ultrasound scanning of rat livers in vitro.

<p>The experimental setup for ultrasound scanning of rat livers in vitro.</p

Overexpression of CTHRC1 in Hepatocellular Carcinoma Promotes Tumor Invasion and Predicts Poor Prognosis

<div><p>Collagen triple helix repeat containing-1 (CTHRC1) is a secreted glycoprotein that activates the planar cell polarity pathway of Wnt signaling. Using microarray analysis, we found that the <i>CTHRC1</i> gene is overexpressed in hepatocellular carcinoma (HCC). The level of <i>CTHRC1</i> mRNA was measured in 201 surgically resected HCCs using real time reverse transcription-polymerase chain reaction. Overexpression of CTHRC1 in HCC was associated with large tumor size and advanced tumor stage. Furthermore, expression of CTHRC1 as was identified as an independent prognostic factors in the multivariate analysis. Suppression of CTHRC1 expression inhibited tumor migration and invasion whereas overexpression of CTHRC1 promoted tumor invasion. Activation of RhoA, but not Rac1 or Cdc42, was found to play a crucial role in CTHRC1-induced cell migration. CTHRC1 promoted adhesion of cancer cells to extracellular matrix through induction of integrin β1 expression and activation of focal adhesion kinase. These results suggest CTHRC1 promotes tumor invasion and metastasis by enhancing the adhesion and migratory abilities of tumor cells. It is also a promising biomarker for predicting the prognosis of patients with HCC.</p></div

p15^PAF expression in normal adult tissues.

<p>Nuclear expression of p15^PAF was detected in the lymphocytes in the germinal center (arrow) of lymph node (B), the epithelial cells in the lower half of crypt (arrow) in the colonic mucosa (D), and the suprabasal keratinocytes (arrow) of epidermis (F), but not in the neurons and glial cells of brain (D), hepatocytes of liver (E), and (F) pneumocytes of lung.</p