ENTREP/FAM189A2 encodes a new ITCH ubiquitin ligase activator that is downregulated in breast cancer.

The HECT-type ubiquitin E3 ligases including ITCH regulate many aspects of cellular function through ubiquitinating various substrates. These ligases are known to be allosterically autoinhibited and to require an activator protein to fully achieve the ubiquitination of their substrates. Here we demonstrate that FAM189A2, a downregulated gene in breast cancer, encodes a new type of ITCH activator. FAM189A2 is a transmembrane protein harboring PPxY motifs, and the motifs mediate its association with and ubiquitination by ITCH. FAM189A2 also associates with Epsin and accumulates in early and late endosomes along with ITCH. Intriguingly, FAM189A2 facilitates the association of a chemokine receptor CXCR4 with ITCH and enhances ITCH-mediated ubiquitination of CXCR4. FAM189A2-knockout prohibits CXCL12-induced endocytosis of CXCR4, thereby enhancing the effects of CXCL12 on the chemotaxis and mammosphere formation of breast cancer cells. In comparison to other activators or adaptors known in the previous studies, FAM189A2 is a unique activator for ITCH to desensitize CXCR4 activity, and we here propose that FAM189A2 be renamed as ENdosomal TRansmembrane binding with EPsin (ENTREP).

Diffuse mesothelin expression leads to worse prognosis through enhanced cellular proliferation in colorectal cancer.

Mesothelin (MSLN) is a glycophosphatidylinositol (GPI)-linked cell surface protein that is highly expressed in several types of malignant tumor, including malignant pleural mesothelioma, ovarian cancer and pancreatic adenocarcinoma. Recently, a comprehensive immunohistochemical study using MN-1 monoclonal antibody identified a significant number of colorectal tumors in which MSLN was expressed. However, the clinicopathological profiles and survival of patients with MSLN-positive colorectal cancer have not been fully analyzed. In the current study, the expression of MSLN in 270 primary and 44 metastatic colorectal tumors was immunohistochemically analyzed to determine the clinical usefulness of MSLN immunohistochemistry and to identify potential candidates for future anti-MSLN therapy. In vitro experiments using colon cancer cell lines were performed to investigate the biological significance of MSLN expression in tumors. The results of univariate analyses identified a significant correlation between MSLN expression and females (P=0.0042). Furthermore, an inverse correlation between MSLN expression and solid/sheet-like proliferation (P=0.014) was also revealed. Additionally, overall survival was significantly shorter in patients with diffuse luminal/membranous expression of MSLN (P=0.018). Multivariable Cox hazards regression analysis revealed diffuse MSLN expression (hazard ratio, 2.26; 95% confidence interval, 1.04-4.91; P=0.039) as a potential risk factor. When comparing primary CRCs and the metastasis of each, a weakly positive correlation was identified for MSLN positivity (% positive cells; R=0.484; P<0.0001). The in vitro experiments revealed a positive role for MSLN in colon cancer cell proliferation. Thus, MSLN immunohistochemistry may be useful in the prognostication of patients with CRC. The results demonstrated that significant numbers of patients with MSLN-positive CRC exhibiting metastasis could be targeted by anti-MSLN therapies.

Indispensable role of STIL in the regulation of cancer cell motility through the lamellipodial accumulation of ARHGEF7-PAK1 complex.

Cell motility is a tightly regulated phenomenon that supports the accurate formation of organ structure during development and homeostasis, including wound healing and inflammation. Meanwhile, cancer cells exhibit dysregulated motility, which causes spreading and invasion. The Dbl family RhoGEF ARHGEF7/ß-PIX and its binding partner p21-activated kinase PAK1 are overexpressed in a variety of cancers and have been shown to be responsible for cancer cell migration. A key step in motility is the intracellular transport of ARHGEF7-PAK1 complex to the migrating front of cells, where lamellipodia protrusion and cytoskeletal remodeling efficiently occur. However, the molecular mechanisms of the intracellular transport of this complex are not fully understood. Here we revealed that SCL/TAL1-interrupting locus (STIL) is indispensable for the efficient migration of cancer cells. STIL forms a ternary complex with ARHGEF7 and PAK1 and accumulates with those proteins at the lamellipodia protrusion of motile cells. Knockdown of STIL impedes the accumulation of ARHGEF7-PAK1 complex within membrane ruffles and attenuates the phosphorylation of PAK1 substrates and cortical actin remodeling at the migrating front. Intriguingly, ARHGEF7 knockdown also diminishes STIL and PAK1 accumulation in membrane ruffles. Either STIL or ARHGEF7 knockdown impedes cell migration and Rac1 activity at the migrating front of cells. These results indicate that STIL is involved in the ARHGEF7-mediated positive-feedback activation of cytoskeletal remodeling through accumulating the ARHGEF7-PAK1 complex in lamellipodia. We conclude that its involvement is crucial for the polarized formation of Rac1-mediated leading edge, which supports the efficient migration of cancer cells.

CD70 expression in tumor-associated fibroblasts predicts worse survival in colorectal cancer patients.

The anticancer effects of immune checkpoint inhibitors against CTLA4 and CD274-PDCD1 axes are evident. However, these immunotherapies for colorectal cancers (CRCs) are now limited to a small subset of patients with microsatellite unstable tumors. Thus, therapeutics targeting other types of CRCs is desired. The CD70-CD27 axis plays a co-stimulatory role in promoting the expansion and differentiation of T-lymphocytes through the activation of NFκB pathway. Aberrant activation of the CD70-CD27 axis accelerates tumor cell proliferation, survival, and immune evasion of tumor cells. Based on these observations, drugs modulating the CD70-CD27 axis have been developed with expectation of anticancer effects. In the present study, 269 primary CRCs were evaluated immunohistochemically for CD70, CD27, and FOXP3 expression to assess their clinical usage and the application of CD70-CD27 axis modulating drugs. CRC tumor cells rarely (2.2%) expressed CD70. In contrast, tumor-surrounding fibroblasts showed various CD70 expressions (fCD70) in 14.9%. The logistic regression analysis revealed significant association of fCD70 expression with incomplete resection status (OR, 2.60; 95% CI, 1.10-6.13; P = 0.029). Overall survival was significantly decreased in the cohort of the patients with fCD70-positive tumor (P = 0.0078). Furthermore, significantly more CD27+ tumor-associated lymphocytes were detected within the primary CRCs without metastases (P = 0.024). Thus, the CD70-CD27 axis may have several roles in CRCs independent from their mismatch repair (MMR) system status. CD70-CD27 pathway-modulating therapies may be applied to CRC patients regardless of their tumor MMR status.

Down-regulation of the zinc-finger homeobox protein TSHZ2 releases GLI1 from the nuclear repressor complex to restore its transcriptional activity during mammary tumorigenesis.

Although breast cancer is one of the most common malignancies, the molecular mechanisms underlying its development and progression are not fully understood. To identify key molecules involved, we screened publicly available microarray datasets for genes differentially expressed between breast cancers and normal mammary glands. We found that three of the genes predicted in this analysis were differentially expressed among human mammary tissues and cell lines. Of these genes, we focused on the role of the zinc-finger homeobox protein TSHZ2, which is down-regulated in breast cancer cells. We found that TSHZ2 is a nuclear protein harboring a bipartite nuclear localization signal, and we confirmed its function as a C-terminal binding protein (CtBP)-dependent transcriptional repressor. Through comprehensive screening, we identified TSHZ2-suppressing genes such as AEBP1 and CXCR4, which are conversely up-regulated by GLI1, the downstream transcription factor of Hedgehog signaling. We found that GLI1 forms a ternary complex with CtBP2 in the presence of TSHZ2 and that the transcriptional activity of GLI1 is suppressed by TSHZ2 in a CtBP-dependent manner. Indeed, knockdown of TSHZ2 increases the expression of AEBP1 and CXCR4 in TSHZ2-expressing immortalized mammary duct epithelium. Concordantly, immunohistochemical staining of mammary glands revealed that normal duct cells expresses GLI1 in the nucleus along with TSHZ2 and CtBP2, whereas invasive ductal carcinoma cells, which does not express TSHZ2, show the increase in the expression of AEBP1 and CXCR4 and in the cytoplasmic localization of GLI1. Thus, we propose that down-regulation of TSHZ2 is crucial for mammary tumorigenesis via the activation of GLI1.

GLI1 orchestrates CXCR4/CXCR7 signaling to enhance migration and metastasis of breast cancer cells.

The up-regulation of chemokine receptors CXCR4 and CXCR7 impacts on the distant metastasis and prognosis of breast cancer, though knowledge about the regulatory mechanism of their expressions is limited. Meanwhile, the GLI transcription factors of Hedgehog signaling have been reported to play a pivotal role in the development and progression of many types of human cancer. In breast cancer, the increased expression of GLI1 correlated with metastasis and unfavorable overall prognosis, though its molecular mechanism is also not fully understood. Based on our findings that GLI1 enhanced the lung metastasis of breast cancer cells in a mouse model system, we comprehensively screened for genes up-regulated by GLI1 in breast cancer cells, and as such identified CXCR4, CXCR7/ACKR3, and actin-binding protein LCP1/L-PLASTIN, all of which have been reported to be involved in CXCL12-stimulating signaling. In breast cancer cells, we found that GLI1 and GLI2 up-regulated these expressions, while treatment with GLI-specific inhibitor GANT61 reduced the expressions. As for CXCR4, we confirmed it as a direct target of GLI1 through the reporter assay and the chromatin immunoprecipitation assay. We also found that GLI1 enhanced CXCL12-induced ERK phosphorylation and cell migration, both of which were blocked by either CXCR4-specific inhibitor or knockdown of CXCR7 or LCP1. These evidences suggest an indispensable role of GLI1 in the migration and metastasis of breast cancer cells through CXCL12/CXCR4 signaling enhancement.