Clinical significance of MYC family protein expression in surgically resected high-grade neuroendocrine carcinoma of the lung.

OBJECTIVES: MYC family genes including MYC, MYCN, and MYCL are amplified and overexpressed as oncogenic drivers in high-grade neuroendocrine carcinoma of the lung (HGNEC), but little is known about their clinical significance. This study evaluated the prognostic impact of MYC family protein expression in patients with surgically resected HGNEC. METHODS: Immunohistochemical analyses were performed on 83 resected specimens of HGNEC using antibodies against MYC family proteins (c-MYC, n-MYC, and l-MYC). When nuclear staining of any intensity in ≥10% of tumor cells showed immunoreactivity with any one or more of c-MYC, n-MYC, or l-MYC, the specimens were defined as MYC family-positive. RESULTS: A total of 83 patients were analyzed. MYC family-positive status was observed in 33.7% (28 of 83 cases) and was not correlated with clinicopathological factors. The protein expression was mutually exclusive and no duplicate cases were observed. A log-rank test showed that MYC family-positive status was significantly associated with shorter overall survival (OS) (p = 0.003) and recurrence-free survival (RFS) (p = 0.039). According to Cox multivariate analysis, MYC family-positive status had a significant effect on shorter OS (hazard ratio [HR] = 2.217, 95% confidence interval [CI] 1.179-4.169, p = 0.014) and RFS (HR = 1.802, 95% CI 1.014-3.202, p = 0.045). In patients with pathological stage I, MYC family-positive status also showed significantly poor OS (HR = 2.847, 95% CI 1.236-6.557, p = 0.014) and RFS (HR = 2.088, 95% CI 1.006-4.332, p = 0.048) in the multivariate analysis. CONCLUSIONS: MYC family protein expression could be an independent unfavorable prognostic factor in patients with surgically resected HGNEC.

High mRNA expression of POU2F3 in small cell lung cancer cell lines predicts the effect of lurbinectedin.

BACKGROUND: Small cell lung cancer (SCLC) is a progressive disease with a poor prognosis. Recently, a method to classify SCLC by the expression status of four transcription factors, ASCL1, NEUROD1, POU2F3, and YAP1, was proposed. Here, we investigated the potential relationships between expression of these four transcription factors and the effect of lurbinectedin. METHODS: mRNA and protein expression of ASCL1, NEUROD1, POU2F3, and YAP1 were quantified in eight SCLC cell lines and analyzed for potential correlations with drug sensitivity. In addition, ASCL1, NEUROD1, POU2F3, and YAP1 expression were evaluated in 105 resected cases of high-grade neuroendocrine carcinoma of the lung, including 59 resected cases of SCLC. RESULTS: Based on the results of qRT-PCR and western blot analyses, the eight SCLC cell lines examined were classified into NEUROD1, POU2F3, and YAP1 subtypes, as well as five ASCL1 subtypes. There were no correlations between cell line subtype classification and drug sensitivity to cisplatin, etoposide, or lurbinectedin. Next, we compared relative mRNA expression levels of each transcription factor with drug sensitivity and found that the higher the mRNA expression level of POU2F3, the lower the IC50 of lurbinectedin. Evaluation of resected SCLC tissue revealed that the composition of subtypes defined by the relative dominance of ASCL1, NEUROD1, POU2F3, and YAP1 was as follows: 61% ASCL1, 15% NEUROD1, 14% POU2F3, 5% YAP1, and 5% all-negative. CONCLUSION: In our experiments, high mRNA expression of POU2F3 in SCLC cell lines correlated with the effect of lurbinectedin.

Investigation of the Subcellular Localization-Dependent Anti- or Pro-Tumor Functions of Maspin in Human Lung Adenocarcinoma Cell Line.

BACKGROUND: Mammary serine protease inhibitor (maspin) is well known as a tumor suppressor gene in several types of cancers and its nuclear localization is essential for its tumor-suppressive function. We previously reported that the cytoplasmic-only localization of maspin is significantly correlated with unfavorable prognosis in patients with lung adenocarcinoma (LUAD). To clarify whether maspin in LUAD acts as a tumor promoter or suppressor, we examined the subcellular localization-dependent biological functions of maspin in human LUAD cell lines. METHODS: The expression levels and subcellular localization of maspin were investigated by performing immunoblotting and immunofluorescence in human LUAD cell lines (PC-9, A549, NCI-H23, RERF-LC-KJ) and human bronchial epithelial cell line (BEAS-2B). We then established stable cell lines overexpressing maspin (A549-maspin and RERF-LC-KJ-maspin) and investigated their subcellular localization. Cell invasion assays of these cell lines were performed to examine their invasiveness. Moreover, the mRNA expression levels between epithelial cell markers (E-cadherin) and mesenchymal cell markers (N-cadherin and vimentin) were compared. RESULTS: The expression of maspin in PC-9 cells was comparable to that in BEAS-2B cells, whereas its expression in A549, NCI-H23, and RERF-LC-KJ cells was decreased. The cell invasion capability of A549-maspin cells showing pancellular expression was significantly decreased compared with that of A549-control cells. By contrast, the cell invasion capability of RERF-LC-KJ-maspin cells showing cytoplasmic-only expression was significantly increased compared with that of RERF-LC-KJ-control cells. The mRNA expression levels of N-cadherin, but not E-cadherin and vimentin, in A549-maspin cells was significantly downregulated compared with that in A549-control cells. No significant differences in these markers were observed between RERF-LC-KJ-maspin and RERF-LC-KJ-control cells. CONCLUSION: The invasive capability of LUAD cells is regulated by the intracellular localization of maspin. Clarification of the molecular mechanism underlying the subcellular localization-dependent function of maspin will promote a deeper understanding of LUAD development and progression.

Gene expression profiling using targeted RNA-sequencing to elucidate the progression from histologically normal lung tissues to non-invasive lesions in invasive lung adenocarcinoma.

Lung adenocarcinoma (LUAD) shows heterogeneous morphological features and the stepwise progression from adenocarcinoma in situ to minimally invasive adenocarcinoma to invasive LUAD. Although multiple genetic alterations have been linked to the progression, the differences between the gene expression profiles of non-invasive lesions (non-ILs) and adjacent histologically normal lung (aNL) tissues within invasive LUAD have not been investigated. Herein, we analyzed differentially expressed genes (DEGs) specific to early-stage carcinogenesis in LUAD. Invasive LUAD tissue samples containing both non-ILs and aNL tissues were obtained from seven patients with pathological stage I LUAD, and each component was subjected to microdissection. Gene expression profiles of each component were determined using targeted RNA-sequencing. In total, 2536 DEGs, including 863 upregulated and 1673 downregulated genes, were identified in non-ILs. In non-ILs, the expression of SLC44A5, a choline transporter-like protein-coding gene, was significantly upregulated, and that of TMEM100, a gene encoding a transmembrane protein, was significantly downregulated. Reportedly, SLC44A5 plays an important role in the development and progression of hepatocellular carcinoma, whereas TMEM100 functions as a tumor suppressor in non-small cell lung cancer. Gene set enrichment analysis showed that DEGs in non-ILs were negatively enriched in cell death and immune response. Immunohistochemical analysis revealed that increased SLC44A5 expression and decreased TMEM100 expression were maintained in ILs. A protein-protein interaction (PPI) network analysis identified several upregulated and downregulated hub genes with high degrees in non-ILs. In conclusion, several new DEGs and key PPI network hub genes were identified in non-ILs, contributing to understanding of early-stage carcinogenesis in LUAD.

Clinical Significance of Serglycin Expression in Human Breast Cancer Patients.

BACKGROUND/AIM: Serglycin plays a crucial role in the aggressiveness of several types of malignancies, including breast cancer. In this study, we aimed to investigate the prognostic impact of serglycin expression in breast cancer patients, which has not been previously reported. PATIENTS AND METHODS: Immunohistochemical analyses were performed on 348 resected specimens of invasive carcinomas, using antibodies against serglycin. RESULTS: Low serglycin expression was observed in 23% of specimens (80/348) and significantly correlated with high histological grade (p=0.001) and negative ER (p=0.013). The log-rank test showed that low serglycin expression correlated with shorter distant metastasis-free survival (DMFS) (p=0.016) and disease-specific survival (DSS) (p=0.037) in node-positive breast cancer patients. Cox's multivariate analysis revealed that low serglycin expression was an independent factor for shorter DMFS (p=0.017) and DSS (p=0.020) in node-positive breast cancer patients. CONCLUSION: Low serglycin expression is an independent predictor of unfavorable prognosis in node-positive breast cancer patients.

Cytoplasmic-only Expression of Maspin Predicts Poor Prognosis in Patients With Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: Maspin has tumor-suppressor functions; however, its prognostic value in patients with oral squamous cell carcinoma (OSCC) remains unknown. We aimed to assess the prognostic importance of the subcellular localization of maspin in patients with OSCC. PATIENTS AND METHODS: Eighty resected specimens were analyzed by immunohistochemistry. Cytoplasmic-only expression observed in >10% of the tumor was defined as maspin-positive. RESULTS: The maspin-positive status (25%) was significantly associated with a higher recurrence rate and shorter disease-free survival (DFS). Cox's multivariate analysis showed that maspin-positive status was an independent factor for shorter DFS. All OSCC cell lines (HSC2, HSC3, HSC4, Ca9-22 and SAS) showed maspin protein localization to both the cytoplasm and nucleus using western blot analysis. In HSC4 cells, cell invasion was significantly increased in response to maspin knockdown. CONCLUSION: Cytoplasmic-only expression of maspin could be an independent poor prognostic factor for patients with OSCC.

Role of cytoplasmic localization of maspin in promoting cell invasion in breast cancer with aggressive phenotype.

Mammary serine protease inhibitor (maspin) is a tumor suppressor gene that is downregulated during carcinogenesis and breast cancer progression. While the nuclear localization of maspin is essential for tumor suppression, we previously reported that the cytoplasmic localization of maspin was significantly correlated with poor prognosis in breast cancer patients. To understand the mechanisms that underlie oncogenic role of cytoplasmic maspin, we studied its biological function in breast cancer cell lines. Subcellular localization of maspin in MDA-MB-231 breast cancer cells was mainly detected in the cytoplasm, whereas in MCF10A mammary epithelial cells, maspin was present in both cytoplasm and nucleus. In MDA-MB-231 cells, maspin overexpression promoted cell proliferation and cell invasion, whereas maspin downregulation resulted in the opposite effect. Further, we observed that SRGN protein levels were increased in MDA-MB-231 cells stably overexpressing maspin. Finally, maspin overexpression in MDA-MB-231 cells resulted in the N-cadherin and epithelial mesenchymal transition (EMT)-related transcription factors upregulation, and TGFß signaling pathway activation. These results suggested that cytoplasmic maspin enhances the invasive and metastatic potential in breast cancer cells with aggressive phenotype by inducing EMT via SRGN/TGFß axis. This study demonstrated a novel biological function of cytoplasmic maspin in progression of breast cancer cells with an aggressive phenotype.

Cytoplasmic-only Expression of Maspin Predicts Unfavorable Prognosis in Patients With Pancreatic Ductal Adenocarcinoma.

BACKGROUND/AIM: Maspin is a tumor-suppressor protein expressed in >90% of pancreatic ductal adenocarcinoma (PDAC) cases. We aimed to assess the prognostic value of subcellular localization of maspin. PATIENTS AND METHODS: Ninety-two resected PDAC specimens were immunohistochemically analyzed. Cytoplasmic-only expression observed in >10% of the tumor was defined as maspin-positive. RESULTS: The maspin-positive status (21.7%) was inversely correlated with well-differentiated histological type and indicated a shorter recurrence-free survival (RFS) and overall survival (OS). Cox's multivariate analysis showed that maspin-positive status was an independent factor for shorter RFS and OS. Maspin was localized to cytoplasm in AsPC-1 cells, but to both nucleus and cytoplasm in BxPC-3 cells. In AsPC-1 cells, cell invasion was significantly reduced in response to maspin suppression via transfection with siRNA targeting maspin, whereas no reduction was observed in BxPC-3 cells. CONCLUSION: Cytoplasmic-only expression of maspin could be an independent unfavorable prognostic indicator for patients with PDAC.

Podoplanin expression in cancer-associated fibroblasts predicts unfavorable prognosis in node-negative breast cancer patients with hormone receptor-positive/HER2 - negative subtype.

BACKGROUND: Podoplanin expression in cancer-associated fibroblasts (CAFs) has been proposed as an indicator for poor prognosis in patients with invasive breast carcinomas, but little is known about its clinical significance in node-negative breast cancer patients with hormone receptor (HR) + /HER2 - subtype, who are expected to have a favorable prognosis. METHODS: Immunohistochemical analyses were performed on 169 resected specimens of node-negative invasive carcinoma of no special type with HR + /HER2 - subtype using antibodies for podoplanin. When more than 10% of CAFs showed immunoreactivity with podoplanin as strong as that of internal positive controls, the specimens were judged as podoplanin-positive. RESULTS: Podoplanin-positive status in CAFs was observed in 16.0% (27 of 169 cases) and it associated with high Ki67 labeling index (LI) (> 30%) (p = 0.03), higher stromal tumor-infiltrating lymphocytes (p < 0.001) and progesterone receptor-negative status (p = 0.045). Log-rank test showed that podoplanin-positive status in CAFs correlated with shorter disease-free survival (DFS) (p = 0.007) and disease-specific survival (DSS) (p < 0.001). Univariate analysis showed a significant correlation between shorter DFS and podoplanin-positive status in CAFs (hazard ratio [HR] = 3.380; p = 0.012), the presence of lymphatic invasion (HR = 5.621; p < 0.001), high Ki67 LI (HR = 5.217; p < 0.001), and histological grade III (HR = 3.748; p = 0.008). According to Cox multivariate analysis, podoplanin-positive status in CAFs had the most significant effect on shorter DSS (HR = 37.759; p = 0.003) followed by high Ki67LI (HR = 27.664; p = 0.007). CONCLUSION: Podoplanin expression in CAFs could be an independent predictor for poor prognosis in node-negative breast cancer patients with HR + /HER2 - subtype.

Gene expression profiling by targeted RNA sequencing in pathological stage I lung adenocarcinoma with a solid component.

OBJECTIVES: Solid predominant adenocarcinoma is considered an independent predictor of an unfavorable prognosis in patients with stage I lung adenocarcinoma (LUAD). Furthermore, solid minor components are related to poor prognosis in patients with stage I LUAD. Therefore, it is imperative to elucidate the molecular determinants of the malignant potential of solid components (SC). Several studies reported the gene expression profiling specific for lepidic predominant adenocarcinoma or solid predominant adenocarcinoma, however; there is no report identifying the differentially expressed genes (DEGs) between SC and acinar component (AC) within the same tumor tissue in pathological (p)-stage I LUAD patients. MATERIALS AND METHODS: LUAD tissue samples containing both SC and AC were obtained from 8 patients with p-stage I LUAD and each component was microdissected. Targeted RNA sequencing was performed by a high-throughput chip-based approach. RESULTS: In total, 1272 DEGs were identified, including 677 upregulated genes and 595 downregulated genes in SC compared with AC. The most highly upregulated gene was TATA binding protein associated factor 7 (TAF7) and the most highly downregulated gene was homeobox B3 (HOXB3), which acts as a metastasis suppressor. A protein-protein interaction (PPI) network analysis of upregulated genes in SC identified ribosomal protein S27a (RPS27a) as a hub gene with the highest degree. First neighbors of RPS27a included PSMA6, which is a highly promising target for lung cancer. The subnetwork of PD-L1 had 10 first neighbors, including CMTM6, which enhances the ability of PD-L1-expressing tumor cells to inhibit T cells. The staining score for PD-L1 in SC was significantly higher than that in AC by immunohistochemistry (p = 0.001). CONCLUSION: Our results revealed several new DEGs and key PPI network in SC compared to AC, contributing to understanding the biological features of SC and providing therapeutic targets for early-stage LUAD with SC in the future.

Increased regulatory B cells are involved in immune evasion in patients with gastric cancer.

Accumulating evidence has indicated that immune regulatory cells are involved in the establishment of tumoral immune evasion. However, the role of regulatory B cells (Bregs) in this remains unclear. Here, we identified a role for Bregs in immune evasion in gastric cancer (GC) patients. The frequency of peripheral Bregs was significantly higher in GC patients than in healthy controls (P = 0.0023). Moreover, the frequency of CD19+CD24hiCD27+ B cells in GC tissue was significantly higher than in peripheral blood and healthy gastric tissue. Carboxyfluorescein succinimidyl ester labeling revealed that CD19+CD24hiCD27+ B cells could suppress the proliferation of autologous CD4+ T cells. Moreover, CD19+CD24hiCD27+ B cells inhibited the production of interferon-gamma by CD4+ T cells. Double staining immunohistochemistry of interleukin-10 and CD19 revealed 5-year overall survival rates of 65.4% and 13.3% in BregLow and BregHigh groups, respectively (P < 0.0001). Multivariate analysis indicated that the frequency of Bregs was an independent prognostic indicator in GC patients. Taken together, our results show the existence of Bregs in GC tissue, and indicate that they are significantly correlated with the prognosis of GC patients.

Upregulation of glucose and amino acid transporters in micropapillary carcinoma.

Micropapillary carcinoma (MPC), a relatively rare histologic carcinoma observed in various organs, is associated with vascular invasion, nodal metastasis, and poor prognosis. MPC is different from papillary carcinoma as it has no fibrovascular core and is thus considered essentially hypovascular. MPCs are known to upregulate glucose transporter 1 (GLUT1) via the activation of a transcription factor, hypoxia-inducible factor (HIF)-1. Here we evaluated the expression of nutrient transporters in MPCs to gain a better understanding of the system used by MPCs to compensate for their intrinsic poor vascularity. We immunohistochemically evaluated 29 MPCs including breast (n=14), lung (n=8), gastrointestinal tract (n=5), and urinary tract cancers (n=2), and compared them with non-micropapillary control cancers (n=32) regarding the expression of amino acid (ASCT1, ASCT2, LAT1, and SNAT1) and glucose (GLUT1, GLUT2) transporters. Each section was scored by the staining intensity (0-3) multiplied by the occupying area (0-10), with a possible range 0-30. The average scores of the MPC and control groups were compared by Student's or Welch's t-test according to the homoscedasticity. The MPC group showed significantly higher scores for ASCT1 (p=0.007), ASCT2 (p=0.001), GLUT1 (p<0.001), and GLUT2 (p<0.001), whereas no significant scores were noted for LAT1 and SNAT1. In conclusion, MPC could be associated with the upregulation of several nutrient transporters, which may contribute to the malignant potential by supporting the survival of cancer cells.

CD44 standard isoform is involved in maintenance of cancer stem cells of a hepatocellular carcinoma cell line.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. Cancer stem cells (CSCs) have attracted attention as a novel therapeutic target for cancer because they play important roles in the development and aggravation of cancer. CD44 is expressed as a standard isoform (CD44s) and several variant isoforms. CD44v is a major isoform expressed on CSCs of a variety of tumors and has been extensively studied. However, HCC tissues dominantly express CD44s, whose function in CSCs remains unclear. In the present study, we investigated the roles of CD44s in CSCs of HCC. Knock-out of the CD44 gene in HuH7 HCC cells on which only CD44s is expressed resulted in decreased spheroid formation and increased drug sensitivity. The expression of CSC marker genes, including CD133 and EpCAM, was significantly downregulated in the spheroids of CD44-deficient cells compared with those in the spheroids of HuH7 cells. In addition, CD44 deficiency impaired antioxidant capacity, concomitant with downregulation of glutathione peroxidase 1 (GPX1) and thioredoxin. Because GPX1 uses the reduced form of glutathione (GSH) to regenerate oxidized cellular components, GSH levels were significantly increased in the CD44-deficient cells. We also found that NOTCH3 and its target genes were downregulated in the spheroids of CD44-deficient cells. NOTCH3 expression in HCC tissues was significantly increased compared with that in adjacent nontumor liver tissues and was correlated with CD44 expression. These results suggest that CD44s is involved in maintenance of CSCs in a HCC cell line, possibly through the NOTCH3 signaling pathway.

Identification of genes involved in the regulation of TERT in hepatocellular carcinoma.

Telomerase reverse transcriptase (TERT) promotes immortalization by protecting telomeres in cancer cells. Mutation of the TERT promoter is one of the most common genetic alterations in hepatocellular carcinoma (HCC), indicating that TERT upregulation is a critical event in hepatocarcinogenesis. Regulators of TERT transcription are, therefore, predicted to be plausible targets for HCC treatment. We undertook a genome-wide shRNA library screen and identified C15orf55 and C7orf43 as regulators of TERT expression in HepG2 cells. Promoter assays showed that C15orf55- and C7orf43-responsive sites exist between base pairs -58 and +36 and -169 and -59 in the TERT promoter, respectively. C15orf55 upregulates TERT expression by binding to two GC motifs in the SP1 binding site of the TERT promoter. C7orf43 upregulates TERT expression through Yes-associated protein 1. The expression levels of C15orf55 and C7orf43 also correlated with that of TERT, and were significantly increased in both HCC tissues and their adjacent non-tumor tissues, compared to normal liver tissues from non-HCC patients. Analysis of 377 HCC patients in The Cancer Genome Atlas dataset showed that overall survival of patients with low levels of C15orf55 and C7orf43 expression in tumor tissues was better compared with patients with high levels of C15orf55 and/or high C7orf43 expression. These results indicate that C15orf55 and C7orf43 are involved in the incidence and progression of HCC by upregulating TERT. In conclusion, we identified C15orf55 and C7orf43 as positive regulators of TERT expression in HCC tissues. These genes are promising targets for HCC treatment.

Clinical Significance of Subcellular Localization of Maspin in Patients with Pathological Stage IA Lung Adenocarcinoma.

BACKGROUND/AIM: Maspin is a tumor-suppressor protein and its prognostic value in lung adenocarcinoma has been reported. However, little is known about the clinical impact of subcellular localization of maspin in early-stage lung adenocarcinoma. We aimed to evaluate the clinical significance of subcellular localization of maspin in patients with pathological stage (p-stage) IA lung adenocarcinoma categorized by the new eighth edition TNM classification. PATIENTS AND METHODS: We immunohistochemically analyzed 181 tissue samples from p-stage IA1 (n=37), IA2 (n=92) and IA3 (n=52) lung adenocarcinomas using antibody for maspin. RESULTS: The 181 cases fell into five predominant subtypes: lepidic (n=32), acinar (n=97), papillary (n=30), solid (n=20) and micropapillary (n=2). The frequencies of maspin staining were: cytoplasmic-only in 24.9%; pancellular (nuclear and cytoplasmic) in 8.8%; nuclear-only in 0.6%; no staining in 65.7%. Cytoplasmic-only staining significantly correlated with high pathological T-classification (p=0.039), lymphatic invasion (p=0.002) and poorer tumor differentiation (p=0.002). The patients were followed-up for 12-151 months (median=74 months), and the cytoplasmic-only staining significantly correlated with shorter disease-free survival (DFS) (p=0.034) and disease-specific survival (DSS) (p=0.036) by log-rank tests. In Cox's multivariate analysis, lymphatic invasion had the most significant effect on shorter DFS and DSS. CONCLUSION: The expression of maspin in the cytoplasm alone could be useful for predicting unfavorable prognoses in patients with p-stage IA lung adenocarcinoma.

Podoplanin expression in cancer-associated fibroblasts predicts unfavourable prognosis in patients with pathological stage IA lung adenocarcinoma.

AIMS: Podoplanin expression in cancer-associated fibroblasts (CAFs) has been proposed as an unfavourable indicator in squamous cell carcinoma of the lung, but little is known about its clinical significance in early-stage lung adenocarcinoma. We evaluated the prognostic impact of podoplanin expression in patients with pathological stage (p-stage) IA lung adenocarcinoma as categorised by the 8th edition of the tumour-node-metastasis classification for lung cancer. METHODS AND RESULTS: Immunohistochemical analyses using anti-podoplanin antibody were performed on resected specimens from 158 patients with p-stage IA lung adenocarcinoma. When more than 10% of cancer cells or CAFs showed immunoreactivity with podoplanin, the specimens were classified as podoplanin-positive. Podoplanin-positive status in cancer cells (n = 8) was not correlated with clinicopathological factors or with patient prognosis. Podoplanin-positive status in CAFs (n = 41) was correlated significantly with poorer tumour differentiation (P < 0.001), the presence of lymphatic invasion (P < 0.001) and high-grade (solid and/or micropapillary) components constituting ≥1% of the entire tumour (P < 0.001). The log-rank test showed that podoplanin-positive status in CAFs was associated significantly with shorter disease-free survival (DFS) (P < 0.001) and disease-specific survival (P = 0.015). In Cox's multivariate analysis, podoplanin-positive status in CAFs had the most significant effect on shorter DFS [hazard ratio (HR) = 4.411, P = 0.004], followed by the presence of high-grade components (HR = 3.581, P = 0.013). CONCLUSIONS: Podoplanin expression in CAFs could be an independent predictor of increased risk of recurrence in patients with p-stage IA lung adenocarcinoma.

Expression of Cancer Stem Cell-associated DKK1 mRNA Serves as Prognostic Marker for Hepatocellular Carcinoma.

BACKGROUND/AIM: Cancer stem cells (CSCs) are associated with prognosis of hepatocellular carcinoma (HCC). In our previous study, we created cDNA microarray databases on the CSC population of human HuH7 cells. In the present study, we identified genes that might serve as prognostic markers of HCC by employing existing databases. MATERIALS AND METHODS: Expressions of glutathione S-transferase pi 1 (GSTP1), lysozyme (LYZ), C-X-C motif chemokine ligand 5 (CXCL5), interleukin-8 (IL8) and dickkopf WNT signaling pathway inhibitor 1 (DKK1), the five most highly expressed genes in the CSC cDNA microarray databases, were examined in 99 patients with HCC by real-time polymerase chain reaction (qRT-PCR), and their clinical significance was analyzed. RESULTS: The Kaplan-Meier analysis showed that both overall and cancer-specific survival were significantly longer in patients with low DKK1 expression than in those with high DKK1 expression. The multivariate analysis revealed that overall survival was negatively associated with albumin and positively associated with alkaline phosphatase (ALP), serosal invasion and stage, and cancer-specific survival was positively associated with ALP, portal vein invasion and DKK1 mRNA. CONCLUSION: Expression of CSC-associated DKK1 mRNA might be an unfavorable prognostic marker for patients with HCC.

Subcellular Localization of Maspin Correlates with Histone Deacetylase 1 Expression in Human Breast Cancer.

BACKGROUND/AIM: Maspin is known to be a tumor suppressor protein. Its nuclear localization and endogenous inhibition of histone deacetylase 1 (HDAC1) are considered crucial for its tumor suppressor activity. However, it remains unclear whether subcellular localization of maspin correlates with HDAC1 expression level in human breast cancer. PATIENTS AND METHODS: Immunohistochemical analyses were performed on 164 resected specimens of invasive breast carcinoma using antibodies for maspin and HDAC1. Subcellular localization of maspin protein and HDAC1 mRNA expression level in two human breast cancer cell lines (MCF7, MDA-MB-231) and mammary epithelial cell line (MCF10) were analyzed by immunofluorescence and quantitative polymerase chain reaction, respectively. RESULTS: The frequency of cytoplasmic-only, pancellular (combined nuclear and cytoplasm) and no staining of maspin were 31%, 14.0% and 55%, respectively. The cytoplasmic-only subgroup showed significantly higher histological grade (p=0.004), negative progesterone receptor status (p=0.003) and shorter disease-free survival compared to the pancellular subgroup (p=0.043). High HDAC1 expression was observed in 60% of cases and was significantly correlated with cytoplasmic-only staining compared to pancellular (p<0.001) or no staining (p=0.004). Immunofluorescence analysis revealed that maspin protein was localized mainly in the cytoplasm in MCF7 and MDA-MB-231 cells, while in both the nucleus and cytoplasm in MCF10A cells. HDAC1 mRNA levels were significantly up-regulated in MCF7 and MDA-MB-231 cells compared to MCF10A cells (p<0.001). CONCLUSION: High HDAC1 expression may contribute to the aggressiveness of human breast cancer with cytoplasmic-only expression of maspin.

A Novel Small-molecule WNT Inhibitor, IC-2, Has the Potential to Suppress Liver Cancer Stem Cells.

BACKGROUND/AIM: The presence of cancer stem cells (CSCs) contributes to metastasis, recurrence, and resistance to chemo/radiotherapy in hepatocellular carcinoma (HCC). The WNT signaling pathway is reportedly linked to the maintenance of stemness of CSCs. In the present study, in order to eliminate liver CSCs and improve the prognosis of patients with HCC, we explored whether small-molecule compounds targeting WNT signaling pathway suppress liver CSCs. MATERIALS AND METHODS: The screening was performed using cell proliferation assay and reporter assay. We next investigated whether these compounds suppress liver CSC properties by using flow cytometric analysis and sphere-formation assays. A mouse xenograft model transplanted with CD44-positive HuH7 cells was used to examine the in vivo antitumor effect of IC-2. RESULTS: In HuH7 human HCC cells, 10 small-molecule compounds including novel derivatives, IC-2 and PN-3-13, suppressed cell viability and WNT signaling activity. Among them, IC-2 significantly reduced the CD44-positive population, also known as liver CSCs, and dramatically reduced the sphere-forming ability of both CD44-positive and CD44-negative HuH7 cells. Moreover, CSC marker-positive populations, namely CD90-positive HLF cells, CD133-positive HepG2 cells, and epithelial cell adhesion molecule-positive cells, were also reduced by IC-2 treatment. Finally, suppressive effects of IC-2 on liver CSCs were also observed in a xenograft model using CD44-positive HuH7 cells. CONCLUSION: The novel derivative of small-molecule WNT inhibitor, IC-2, has the potential to suppress liver CSCs and can serve as a promising therapeutic agent to improve the prognosis of patients with HCC.

CD117 expression is a predictive marker for poor prognosis in patients with non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) accounts for >85% of incidences of lung cancer, for which the predicted 5-year survival rates are low and recurrence rates remain high. Although it has been reported that the patients with SCLC cells that possess the cluster of differentiation (CD) 117 marker exhibited poor prognosis and poor response to chemotherapy, no studies concerning the association of CD117 expression with prognosis of the patients with NSCLC have been reported. An in vitro study reportedly revealed that CD117-positive cell populations in NSCLC cell lines exhibited cancer stem cell (CSC) phenotypes including self-renewal and chemoresistance. Therefore, the present study hypothesized that if CD117-positive cells are CSC-like cells, CD117 positivity may be associated with the prognosis of patients with NSCLC. To confirm this hypothesis, the association between CD117 expression in patients with NSCLC and clinicopathological characteristics was investigated. CD177 expression was examined by immunohistochemistry in 99 patients with NSCLC who underwent curative surgical resection. Tumor samples in the present study included 73 samples of adenocarcinoma and 26 of squamous carcinoma. The associations of CD177 expression with clinicopathological features and prognosis were examined. The lymph node metastasis and rates of recurrence were significantly associated with overall survival rates through multivariate analysis (P<0.001 and P<0.001), respectively. A Kaplan-Meier analysis for relapse-free survival and the log-rank test revealed that the patients with CD117-positive cell populations exhibited shorter relapse-free survival rates compared with patients whose cells were CD117-negative (P=0.014). The multivariate analysis demonstrated that venous invasion, pathological stage, and CD117 expression were independent prognostic parameters for relapse-free survival in patients with NSCLC (P=0.001, P=0.001 and P=0.002), respectively. In conclusion, these data suggest that CD117 expression in NSCLC may serve as a useful marker for predicting the prognosis of patients with NSCLC.