CAMK2D: a novel molecular target for BAP1-deficient malignant mesothelioma.

Malignant mesothelioma (MMe) is a rare but aggressive malignancy. Although the molecular genetics of MMe is known, including BRCA1-associated protein-1 (BAP1) gene alterations, the prognosis of MMe patients remains poor. Here, we generated BAP1 knockout (BAP1-KO) human mesothelial cell clones to develop molecular-targeted therapeutics based on genetic alterations in MMe. cDNA microarray and quantitative RT-PCR (qRT-PCR) analyses revealed high expression of a calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D) gene in the BAP1-KO cells. CAMK2D was highly expressed in 70% of the human MMe tissues (56/80) and correlated with the loss of BAP1 expression, making it a potential diagnostic and therapeutic target for BAP1-deficient MMe. We screened an anticancer drugs library using BAP1-KO cells and successfully identified a CaMKII inhibitor, KN-93, which displayed a more potent and selective antiproliferative effect against BAP1-deficient cells than cisplatin or pemetrexed. KN-93 significantly suppressed the tumor growth in mice xenografted with BAP1-deficient MMe cells. This study is the first to provide a potential molecular-targeted therapeutic approach for BAP1-deficient MMe.

Motor neuron TDP-43 proteinopathy in progressive supranuclear palsy and corticobasal degeneration.

TDP-43 is mislocalized from the nucleus and aggregates within the cytoplasm of affected neurons in cases of amyotrophic lateral sclerosis. TDP-43 pathology has also been found in brain tissues under non-amyotrophic lateral sclerosis conditions, suggesting mechanistic links between TDP-43-related amyotrophic lateral sclerosis and various neurological disorders. This study aimed to assess TDP-43 pathology in the spinal cord motor neurons of tauopathies. We examined 106 spinal cords from consecutively autopsied cases with progressive supranuclear palsy (n = 26), corticobasal degeneration (n = 12), globular glial tauopathy (n = 5), Alzheimer's disease (n = 21) or Pick's disease (n = 6) and neurologically healthy controls (n = 36). Ten of the progressive supranuclear palsy cases (38%) and seven of the corticobasal degeneration cases (58%) showed mislocalization and cytoplasmic aggregation of TDP-43 in spinal cord motor neurons, which was prominent in the cervical cord. TDP-43 aggregates were found to be skein-like, round-shaped, granular or dot-like and contained insoluble C-terminal fragments showing blotting pattern of amyotrophic lateral sclerosis or frontotemporal lobar degeneration. The lower motor neurons also showed cystatin-C aggregates, although Bunina bodies were absent in haematoxylin-eosin staining. The spinal cord TDP-43 pathology was often associated with TDP-43 pathology of the primary motor cortex. Positive correlations were shown between the severities of TDP-43 and four-repeat (4R)-tau aggregates in the cervical cord. TDP-43 and 4R-tau aggregates burdens positively correlated with microglial burden in anterior horn. TDP-43 pathology of spinal cord motor neuron did not develop in an age-dependent manner and was not found in the Alzheimer's disease, Pick's disease, globular glial tauopathy and control groups. Next, we assessed SFPQ expression in spinal cord motor neurons; SFPQ is a recently identified regulator of amyotrophic lateral sclerosis/frontotemporal lobar degeneration pathogenesis, and it is also reported that interaction between SFPQ and FUS regulates splicing of MAPT exon 10. Immunofluorescent and proximity-ligation assays revealed altered SFPQ/FUS-interactions in the neuronal nuclei of progressive supranuclear palsy, corticobasal degeneration and amyotrophic lateral sclerosis-TDP cases but not in Alzheimer's disease, Pick's disease and globular glial tauopathy cases. Moreover, SFPQ expression was depleted in neurons containing TDP-43 or 4R-tau aggregates of progressive supranuclear palsy and corticobasal degeneration cases. Our results indicate that progressive supranuclear palsy and corticobasal degeneration may have properties of systematic motor neuron TDP-43 proteinopathy, suggesting mechanistic links with amyotrophic lateral sclerosis-TDP. SFPQ dysfunction, arising from altered interaction with FUS, may be a candidate of the common pathway.

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).

Expression and Prognostic Significance of CD47-SIRPA Macrophage Checkpoint Molecules in Colorectal Cancer.

Despite the confirmed anti-cancer effects of T-cell immune checkpoint inhibitors, in colorectal cancer (CRC) they are only effective in a small subset of patients with microsatellite-unstable tumors. Thus, therapeutics targeting other types of CRCs or tumors refractory to T-cell checkpoint inhibitors are desired. The binding of aberrantly expressed CD47 on tumor cells to signal regulatory protein-alpha (SIRPA) on macrophages allows tumor cells to evade immune destruction. Based on these observations, drugs targeting the macrophage checkpoint have been developed with the expectation of anti-cancer effects against T-cell immune checkpoint inhibitor-refractory tumors. In the present study, 269 primary CRCs were evaluated immunohistochemically for CD47, SIRPA, CD68, and CD163 expression to assess their predictive utility and the applicability of CD47-SIRPA axis-modulating drugs. Thirty-five percent of the lesions (95/269) displayed CD47 expression on the cytomembrane of CRC cells. CRCs contained various numbers of tumor-associated immune cells (TAIs) with SIRPA, CD68, or CD163 expression. The log-rank test revealed that patients with CD47-positive CRCs had significantly worse survival than CD47-negative patients. Multivariate Cox hazards regression analysis identified tubular-forming histology (hazard ratio (R) = 0.23), age < 70 years (HR = 0.48), and high SIRPA-positive TAI counts (HR = 0.55) as potential favorable factors. High tumor CD47 expression (HR = 1.75), lymph node metastasis (HR = 2.26), and peritoneal metastasis (HR = 5.80) were cited as potential independent risk factors. Based on our observations, CD47-SIRPA pathway-modulating therapies may be effective in patients with CRC.

High phospho-histone H3 expression uniquely predicts favorable survival among four markers of cellular proliferation in colorectal cancer.

Colorectal cancer (CRC) is one of the most frequent gastrointestinal cancers worldwide, with high morbidity and mortality rates. Despite numerous attempts to identify prognostic markers for the CRC patients, the significance of the association of cellular proliferation markers with survival is controversial. Here we used immunohistochemistry to detect four markers of cellular proliferation expressed in primary CRC tissue specimens (n = 269) to assess their potential to serve as prognostic factors. CRC cells variably expressed phospho-histone H3 (PHH3) (range, 0-76 per high-powered field (HPF); median, 7 per HPF), cyclin A (CCNA) (range, 11.3-73.7%; median, 32%), geminin (GMNN) (range, 7.8-82.0%; median, 37.1%), and marker of proliferation Ki-67 (MKI67) (range, 4.9-96.6%; median, 49.6%). Among them, patients with PHH3-high (≥7 per HPF) tumors uniquely experienced significantly longer 5-year survival than those with PHH3-low (≤6 per HPF) (81.8% vs. 65.5%; P = 0.0047). Multivariable Cox hazards regression analysis identified PHH3-high (hazard ratio, 0.54; 95% confidence interval, 0.31-0.92; P = 0.025) as potential favorable factors. PHH3 levels inversely associated with pT stage (P < 0.0001) and were significantly and inversely associated with tumor diameter (ρ = -0.314, P < 0.0001). These findings support the use of PHH3 immunohistochemistry for predicting the prognoses of patients with CRC.

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.

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.

Phase I study of concurrent vinorelbine and radiation therapy in high-risk postmastectomy breast cancer patients.

BACKGROUND: Postmastectomy chest wall irradiation is recommended for high-risk breast cancer patients, such as those with ≥4 positive nodes. Irradiation is performed sequentially rather than concurrently with chemotherapy. However, the 5-year locoregional recurrence-free survival was statistically better in the concurrent method in node-positive patients in a prior study. The benefit of concurrent chemoradiotherapy for postmastectomy breast cancer patients is uncertain. Vinorelbine is often used as concurrent chemoradiotherapy for non-small cell lung cancer in Japan and has antitumor activity in breast cancer as well. Thus, we planned this dose-finding study of concurrent vinorelbine and radiation therapy in high-risk postmastectomy breast cancer patients. METHODS: High-risk postmastectomy breast cancer patients were recruited. Patients received weekly vinorelbine administered concurrently with radiation therapy. The radiation dose was 50 Gy in 25 fractions over 5 weeks. Vinorelbine was administered weekly without a break, so the maximum number of vinorelbine cycles was five. A 3 + 3 dose-escalation design was used for determining maximal tolerable dose, recommended dose and safety. RESULTS: A total of 10 patients were enrolled in cohorts of 10 and 15 mg/m(2). Dose-limiting toxicity was observed in one case in 10 mg/m(2) and two cases in 15 mg/m(2). Therefore, the maximal tolerable dose was defined at 15 mg/m(2) and the recommended dose was determined at 10 mg/m(2). The main adverse events included radiation dermatitis and neutropenia. Recurrence was observed in one patient with a median follow-up of 40 months. CONCLUSIONS: Concurrent vinorelbine and radiation therapy has a manageable safety profile at 10 mg/m(2) in high-risk postmastectomy breast cancer patients.

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.

Addiction of pancreatic cancer cells to zinc-finger transcription factor ZIC2.

Activity of GLI transcription factors of Hedgehog signaling is key for various cancer cell properties, especially in pancreatic ductal adenocarcinoma (PDAC). Zinc-finger transcriptional regulators ZIC1 to ZIC5 of ZIC gene family were demonstrated to associate with GLI to increase the nuclear accumulation and transcriptional activity of GLI. Notwithstanding this supportive role for GLI-dependent transcription, it was not fully understood whether ZIC plays an independent role in cancer cell biology. Here, we found that ZIC2 is indispensable in the regulation of PDAC cell apoptosis. We found that human PDAC cell lines uniquely express ZIC2. ZIC2 knockdown induced PDAC cell apoptosis; conversely, ZIC2 over-expression enhanced the cellular proliferation. Through a comprehensive screening, we identified fibroblast growth factor receptor 3 (FGFR3) and ANNEXIN A8 (ANXA8) as genes up-regulated by ZIC2 in PDAC cells. The forced expression of these two genes cooperatively rescued the apoptosis of ZIC2-knockdown cells. Immunohistochemical analyses further supported the correlation of ZIC2 expression and these genes in human pancreata harboring PDAC. Intriguingly, the ZIC2-mediated up-regulation of FGFR3 and ANXA8 was indicated to be GLI -independent. This evidence highlights the indispensable role of ZIC2 in regulating cellular proliferation and apoptosis during PDAC development and suggests a potential therapeutic target for PDAC.

GLI1 interferes with the DNA mismatch repair system in pancreatic cancer through BHLHE41-mediated suppression of MLH1.

The mismatch repair (MMR) system is indispensable for the fidelity of DNA replication, the impairment of which predisposes to the development and progression of many types of cancers. To date, GLI1 transcription factor, a key molecule of the Hedgehog signaling pathway, has been shown to regulate the expression of several genes crucial for a variety of cancer cell properties in many types of cancers, including pancreatic ductal adenocarcinoma (PDAC), but whether GLI1 could control the MMR system was not known. Here, we showed that GLI1 and GLI2 indirectly suppressed the expression of MLH1 in PDAC cells. Through GLI1 target gene screening, we found that GLI1 and GLI2 activated the expression of a basic helix-loop-helix type suppressor BHLHE41/DEC2/SHARP1 through a GLI-binding site in the promoter. Consistent with a previous report that BHLHE41 suppresses the MLH1 promoter activity, we found that the activation of GLI1 led to the BHLHE41-dependent suppression of MLH1, and a double knockdown of GLI1 and GLI2 conversely increased the MLH1 protein in PDAC cells. Using TALEN-based modification of the MLH1 gene, we further showed that GLI1 expression was indeed associated with an increased tolerance to a methylating agent, methylnitrosourea cooperatively with a lower copy number status of MLH1. Finally, GLI1 expression was immunohistochemically related positively with BHLHE41 and inversely with MLH1 in PDAC cells and precancerous lesions of the pancreas. On the basis of these results, we propose that GLI1 depresses the MMR activity and might contribute to the development and progression of PDAC.