Clinicopathological Features and Molecular Signatures of Lateral Neck Lymph Node Metastasis in Papillary Thyroid Microcarcinoma.

BACKGRUOUND: The predictive factors for lateral neck lymph node metastasis (LLNM) in papillary thyroid microcarcinoma (PTMC) remain undetermined. This study investigated the clinicopathological characteristics, transcriptomes, and tumor microenvironment in PTMC according to the LLNM status. We aimed to identify the biomarkers associated with LLNM development. METHODS: We retrospectively reviewed the medical records of patients with PTMC from two independent institutions between 2018 and 2022 (n=597 and n=467). We compared clinicopathological features between patients without lymph node metastasis (N0) and those with LLNM (N1b). Additionally, laser capture microdissection and RNA sequencing were performed on primary tumors from both groups, including metastatic lymph nodes from the N1b group (n=30; 20 primary tumors and 10 paired LLNMs). We corroborated the findings using RNA sequencing data from 16 BRAF-like PTMCs from The Cancer Genome Atlas. Transcriptomic analyses were validated by immunohistochemical staining. RESULTS: Clinicopathological characteristics, such as male sex, multifocality, extrathyroidal extension, lymphatic invasion, and central node metastasis showed associations with LLNM in PTMCs. Transcriptomic profiles between the N0 and N1b PTMC groups were similar. However, tumor microenvironment deconvolution from RNA sequencing and immunohistochemistry revealed an increased abundance of tumor-associated macrophages, particularly M2 macrophages, in the N1b group. CONCLUSION: Patients with PTMC who have a male sex, multifocality, extrathyroidal extension, lymphatic invasion, and central node metastasis exhibited an elevated risk for LLNM. Furthermore, infiltration of M2 macrophages in the tumor microenvironment potentially supports tumor progression and LLNM in PTMCs.

Predictive biomarkers for the responsiveness of recurrent glioblastomas to activated killer cell immunotherapy.

BACKGROUND: Recurrent glioblastoma multiforme (GBM) is a highly aggressive primary malignant brain tumor that is resistant to existing treatments. Recently, we reported that activated autologous natural killer (NK) cell therapeutics induced a marked increase in survival of some patients with recurrent GBM. METHODS: To identify biomarkers that predict responsiveness to NK cell therapeutics, we examined immune profiles in tumor tissues using NanoString nCounter analysis and compared the profiles between 5 responders and 7 non-responders. Through a three-step data analysis, we identified three candidate biomarkers (TNFRSF18, TNFSF4, and IL12RB2) and performed validation with qRT-PCR. We also performed immunohistochemistry and a NK cell migration assay to assess the function of these genes. RESULTS: Responders had higher expression of many immune-signaling genes compared with non-responders, which suggests an immune-active tumor microenvironment in responders. The random forest model that identified TNFRSF18, TNFSF4, and IL12RB2 showed a 100% accuracy (95% CI 73.5-100%) for predicting the response to NK cell therapeutics. The expression levels of these three genes by qRT-PCR were highly correlated with the NanoString levels, with high Pearson's correlation coefficients (0.419 (TNFRSF18), 0.700 (TNFSF4), and 0.502 (IL12RB2)); their prediction performance also showed 100% accuracy (95% CI 73.54-100%) by logistic regression modeling. We also demonstrated that these genes were related to cytotoxic T cell infiltration and NK cell migration in the tumor microenvironment. CONCLUSION: We identified TNFRSF18, TNFSF4, and IL12RB2 as biomarkers that predict response to NK cell therapeutics in recurrent GBM, which might provide a new treatment strategy for this highly aggressive tumor.

miR-22-3p and miR-30e-5p Are Associated with Prognosis in Cervical Squamous Cell Carcinoma.

Alteration in expression of miRNAs can cause various malignant changes and the metastatic process. Our aim was to identify the miRNAs involved in cervical squamous cell carcinoma (SqCC) and metastasis, and to test their utility as indicators of metastasis and survival. Using microarray technology, we performed miRNA expression profiling on primary cervical SqCC tissue (n = 6) compared with normal control (NC) tissue and compared SqCC that had (SqC-M; n = 3) and had not (SqC-NM; n = 3) metastasized. Four miRNAs were selected for validation by qRT-PCR on 29 SqC-NM and 27 SqC-M samples, and nine metastatic lesions (ML-SqC), from a total of 56 patients. Correlation of miRNA expression and clinicopathological parameters was analyzed to evaluate the clinical impact of candidate miRNAs. We found 40 miRNAs differentially altered in cervical SqCC tissue: 21 miRNAs were upregulated and 19 were downregulated (≥2-fold, p < 0.05). Eight were differentially altered in SqC-M compared with SqC-NM samples: four were upregulated (miR-494, miR-92a-3p, miR-205-5p, and miR-221-3p), and four were downregulated (miR-574-3p, miR-4769-3p, miR-1281, and miR-1825) (≥1.5-fold, p < 0.05). MiR-22-3p might be a metastamiR, which was gradually further downregulated in SqC-NM > SqC-M > ML-SqC. Downregulation of miR-30e-5p significantly correlated with high stage, lymph node metastasis, and low survival rate, suggesting an independent poor prognostic factor.

NOX2-Induced High Glycolytic Activity Contributes to the Gain of COL5A1-Mediated Mesenchymal Phenotype in GBM.

The alteration of the cellular metabolism is a hallmark of glioma. The high glycolytic phenotype is a critical factor in the pathogenesis of high-grade glioma, including glioblastoma multiforme (GBM). GBM has been stratified into three subtypes as the proneural, mesenchymal, and classical subtypes. High glycolytic activity was found in mesenchymal GBM relative to proneural GBM. NADPH oxidase 2 (NOX2) has been linked to cellular metabolism and epithelial-mesenchymal transition (EMT) in tumors. The role of NOX2 in the regulation of the high glycolytic phenotype and the gain of the mesenchymal subtype in glioma remain unclear. Here, our results show that the levels of NOX2 were elevated in patients with GBM. NOX2 induces hexokinase 2 (HK2)-dependent high glycolytic activity in U87MG glioma cells. High levels of NOX2 are correlated with high levels of HK2 and glucose uptake in patients with GBM relative to benign glioma. Moreover, NOX2 increases the expression of mesenchymal-subtype-related genes, including COL5A1 and FN1 in U87MG glioma cells. High levels of NOX2 are correlated with high levels of COL5A1 and the accumulation of extracellular matrix (ECM) in patients with GBM relative to benign glioma. Furthermore, high levels of HK2 are correlated with high levels of COL5A1 in patients with GBM relative to benign glioma. Our results suggest that NOX2-induced high glycolytic activity contributes to the gain of the COL5A1-mediated mesenchymal phenotype in GBM.

miR-542-3p Contributes to the HK2-Mediated High Glycolytic Phenotype in Human Glioma Cells.

(1) Background: The elevation of glucose metabolism is linked to high-grade gliomas such as glioblastoma multiforme (GBM). The high glycolytic phenotype is associated with cellular proliferation and resistance to treatment with chemotherapeutic agents in GBM. MicroRNA-542-3p (miR-542-3p) has been implicated in several tumors including gliomas. However, the role of miR-542-3p in glucose metabolism in human gliomas remains unclear; (2) Methods: We measured the levels of cellular proliferation in human glioma cells. We measured the glycolytic activity in miR-542-3p knockdown and over-expressed human glioma cells. We measured the levels of miR-542-3p and HK2 in glioma tissues from patients with low- and high-grade gliomas using imaging analysis; (3) Results: We show that knockdown of miR-542-3p significantly suppressed cellular proliferation in human glioma cells. Knockdown of miR-542-3p suppressed HK2-induced glycolytic activity in human glioma cells. Consistently, over-expression of miR-542-3p increased HK2-induced glycolytic activity in human glioma cells. The levels of miR-542-3p and HK2 were significantly elevated in glioma tissues of patients with high-grade gliomas relative to that in low-grade gliomas. The elevation of HK2 levels in patients with high-grade gliomas were positively correlated with the high levels of miR-542-3p in GBM and low-grade gliomas (LGG) based on the datasets from the Cancer Genome Atlas (TCGA) database. Moreover, the high levels of miR-542-3p were associated with poor survival rate in the TCGA database; (4) Conclusions: miR-542-3p contributes to the HK2-mediated high glycolytic phenotype in human glioma cells.

MicroRNA signatures associated with lymph node metastasis in intramucosal gastric cancer.

Although a certain proportion of intramucosal carcinomas (IMCs) of the stomach does metastasize, the majority of patients are currently treated with endoscopic resection without lymph node dissection, and this potentially veils any existing metastasis and may put some patients in danger. In this regard, biological markers from the resected IMC that can predict metastasis are warranted. Here, we discovered unique miRNA expression profiles that consist of 21 distinct miRNAs that are specifically upregulated (miR-628-5p, miR-1587, miR-3175, miR-3620-5p, miR-4459, miR-4505, miR-4507, miR-4720-5p, miR-4742-5p, and miR-6779-5p) or downregulated (miR-106b-3p, miR-125a-5p, miR-151b, miR-181d-5p, miR-486-5p, miR-500a-3p, miR-502-3p, miR-1231, miR-3609, and miR-6831-5p) in metastatic (M)-IMC compared to nonmetastatic (N)-IMC, or nonneoplastic gastric mucosa. Intriguingly, most of these selected miRNAs showed stepwise increased or decreased expression from nonneoplastic tissue to N-IMC to M-IMC. This suggests that common oncogenic mechanisms are gradually intensified during the metastatic process. Using a machine-learning algorithm, we demonstrated that such miRNA signatures could distinguish M-IMC from N-IMC. Gene ontology and pathway analysis revealed that TGF-ß signaling was enriched from upregulated miRNAs, whereas E2F targets, apoptosis-related, hypoxia-related, and PI3K/AKT/mTOR signaling pathways, were enriched from downregulated miRNAs. Immunohistochemical staining of samples from multiple institutions indicated that PI3K/AKT/mTOR pathway components, MAPK1, phospho-p44/42 MAPK, and pS6 were highly expressed and the expression of SMAD7, a TGF-ß pathway component, was decreased in M-IMC, which could aid in distinguishing M-IMC from N-IMC. The miRNA signature discovered in this study is a valuable biological marker for identifying metastatic potential of IMCs, and provides novel insights regarding the metastatic progression of IMC.

Minimal deviation adenocarcinoma (adenoma malignum) of the uterine cervix: clinicopathological analysis of 17 cases.

OBJECTIVE: The aim of this study was to evaluate the clinicopathological features of minimal deviation adenocarcinoma (MDA) and to analyze its prognostic factors. METHODS: We retrospectively analyzed the medical records of 17 patients who were diagnosed with MDA at a single institution between January 2005 and December 2015. RESULTS: The median age of the patients was 47.7 years (33-75 years). MDA was diagnosed in 7 patients (41.2%) before performing definitive surgery. Stage IB disease was diagnosed in 12 patients (70.6%) and advanced stage disease (stage II: 3, stage III: 2) in 5. MDA was incidentally diagnosed following hysterectomy for benign conditions in 6 patients. Adjuvant therapy was administered to 13 patients (76.5%). During median follow-up over 33.6 months (7-99 months), 11 patients (64.7%) showed no evidence of disease, 6 (35.3%) showed persistent or recurrent disease and 5 died of the disease. Peutz-Jeghers syndrome was not suspected in any patient, and no mutation was detected in the 3 patients who underwent genetic testing. Univariate analysis showed that advanced stage disease (P=0.016) and lymphovascular space invasion (P=0.002) demonstrated a statistically significant association with poor overall survival (OS) rates. Advanced stage disease continued to show a significant association with poor OS rates (hazard ratio, 2.92; 95% confidence interval, 1.097-7.746; P=0.032) even after multivariate analysis. CONCLUSION: Early diagnosis is important to manage MDA. Clinicians should consider MDA among the differential diagnoses in patients with a suspicious clinical presentation even with negative cervical screening tests.

MicroRNA-136 inhibits cancer stem cell activity and enhances the anti-tumor effect of paclitaxel against chemoresistant ovarian cancer cells by targeting Notch3.

To identify microRNAs (miRNAs) regulating Notch3 expression in association with paclitaxel resistance, candidate miRNAs targeting Notch3 were predicted using TargetScan. We found that miR-136 directly targets Notch3, and miR-136 was significantly downregulated in OSC tissues relative to normal control tissues, and low expression of miR-136 correlated with poor overall in ovarian cancer patients. Artificial miR-136 overexpression significantly reduced cell viability, proliferation, Cancer stem cell (CSC) spheroid formation, and angiogenesis, and increased apoptosis in paclitaxel-resistant SKpac cells compared with the effects of paclitaxel alone. miR-136 overexpression downregulated cell survival- (survivin, DNA-PK, pS6, S6) and cell cycle- (Cyclin D1, NF-κB) related proteins, and anti-apoptotic proteins (BCL2, and BCL-XL), and upregulated pro-apoptotic proteins (Bim, Bid, and Bax). Taken together, miR-136 targets the Notch3 oncogene and functions as a tumor suppressor. miR-136 overexpression resensitized paclitaxel-resistant ovarian cancer cells and reduced CSC activities, suggesting a promising new target for the treatment of chemoresistant ovarian cancers.

Sox10 expression in ovarian epithelial tumors is associated with poor overall survival.

Sox10 is a transcription factor regulating the development of several cell lineages and is involved in tumor development. However, the clinicopathological relevance of Sox10 expression in ovarian cancer has not been examined. We assessed expression of Sox10 in ovarian epithelial tumors by immunohistochemistry and assessed its prognostic value by analyzing the correlation between its expression and clinicopathological factors. We used tissue microarrays including 244 ovarian epithelial tumors. Sox10 staining was found in the cytoplasm or nucleus of tumor cells. Malignant serous, mucinous, and endometrioid tumors were significantly more likely to express Sox10 than benign and borderline tumors. Expression patterns in adenocarcinomas were different for histologic subtypes: nuclear Sox10 staining was common in clear-cell adenocarcinomas and serous adenocarcinomas, whereas all cases of mucinous and endometrioid tumors were negative for nuclear staining. Nuclear Sox10 staining was also associated with chemoresistance and shorter overall survival in ovarian adenocarcinomas, notably in high-grade serous adenocarcinoma. Sox10 is expressed in many ovarian carcinomas, suggesting that it might be involved in oncogenesis of ovarian carcinoma. Expression pattern of Sox10 differs between histological subtypes. Nuclear Sox10 expression is an independent indicator of poor prognosis in ovarian adenocarcinomas, notably in high-grade serous adenocarcinomas.

Spermatic Cord Paraganglioma With Histologically Malignant Features.

Paragangliomas occur extremely rarely in the spermatic cord. A 40-year-old man presented with a scrotal mass that was diagnosed as spermatic cord paraganglioma with malignant histological features. To our knowledge, this is the first case of spermatic cord paraganglioma presented with malignant histological features evaluated by histological scoring. Careful evaluation of histological features and systematic evaluation should be considered for patients with spermatic cord paragangliomas.

Germline Mutations of BRCA1 and BRCA2 in Korean Ovarian Cancer Patients: Finding Founder Mutations.

OBJECTIVES: To investigate and analyze the BRCA mutations in Korean ovarian cancer patients with or without family history and to find founder mutations in this group. METHODS/MATERIALS: One hundred two patients who underwent a staging operation for pathologically proven epithelial cancer between January 2013 and December 2014 were enrolled. Thirty-two patients declined to analyze BRCA1/2 gene alterations after genetic counseling and pedigree analysis. Lymphocyte specimens from peripheral blood were assessed for BRCA1/2 by direct sequencing. RESULTS: BRCA genetic test results of 70 patients were available. Eighteen BRCA1/2 mutations and 17 unclassified variations (UVs) were found. Five of the BRCA1/2 mutations and 4 of the UVs were not reported in the Breast Cancer Information Core database. One BRCA2 UV (8665_8667delGGA) was strongly suspicious to be a deleterious mutation. BRCA1/2 mutations were identified in 11 (61.1%) of 18 patients with a family history and in 7 (13.5%) of 52 patients without a family history.Candidates for founder mutations in Korean ovarian cancer patients were assessed among 39 BRCA1/2 mutations from the present study and from literature reviews. The analysis showed that 1041_1043delAGCinsT (n = 4; 10.2%) and 3746insA (n = 4; 10.2%) were possible BRCA1 founder mutations. Only one of the BRCA2 mutations (5804_5807delTTAA) was repeated twice (n = 2; 5.1%). CONCLUSIONS: The prevalence of BRCA1/2 mutations in Korean ovarian cancer patients irrespective of the family history was significantly higher than previously reported. Possible founder mutations in Korean ovarian cancer patients were identified.

VAV3 Overexpressed in Cancer Stem Cells Is a Poor Prognostic Indicator in Ovarian Cancer Patients.

Ovarian carcinoma is a highly lethal malignancy due to frequent relapse and drug resistance. Cancer stem cells (CSCs) are thought to contribute significantly to disease relapse and drug resistance. In this study, a subpopulation of CSCs of ovarian carcinoma was isolated and the genes differentially expressed in these cells were identified to characterize CSCs and to find candidate biomarkers. Ovarian carcinoma cells from patients were primarily cultured, and spheroid-forming cells (SFCs) were isolated. The characteristic genes of SFCs were identified through cDNA microarray and validation by quantitative real-time polymerase chain reaction and immunohistochemistry, and the association of their expression with clinicopathologic parameters was analyzed. GSC (4.26-fold), VAV3 (7.05-fold), FOXA2 (12.06-fold), LEF1 (17.26-fold), COMP (21.33-fold), GRIN2A (9.36-fold), CD86 (23.14-fold), PYY (4.18-fold), NKX3-2 (10.35-fold), and PDK4 (74.26-fold) were significantly upregulated in SFCs compared with parental cancer cells. With validation for human ovarian carcinomas, LEF1, PYY, NKX3-2, and WNT3A were significantly upregulated in chemoresistant cancers compared with chemosensitive cancers. Overexpression of LEF1, VAV3, and NKX3-2 was significantly associated with distant metastasis by immunohistochemistry. VAV3 overexpression was an independent poor survival indicator (hazard ratio=15.27, P<0.05) by multivariate Cox analysis. The further functional assay revealed that VAV3 knockdown regulated CSC activation and ovarian cancer cell proliferation and sensitized paclitaxel (PTX)-resistant cancer cells to PTX treatment. Taken together, we identified by high-throughput analysis of CSCs that VAV3 overexpression is a novel biomarker for poor prognosis and survival in ovarian carcinoma.

Human papillomavirus type-16 positive endobronchial metastasis from uterine cervical cancer.

Although the lung is the most common site of metastasis from extrapulmonary malignancies, endobronchial metastases (EBM) are relatively rare. EBM typically originate from breast, colorectal, or kidney cancer. EBM from uterine cervical cancer is relatively rare and is difficult to confirm. In this study, we report a case of EBM in a patient with previously treated uterine cervical cancer. In this case, differentiation of the EBM from primary bronchogenic carcinoma with clinical, radiological, and pathologic findings was difficult. As identical human papillomavirus (HPV)-16 DNA was detected in both the EBM and in previously resected tissues from the prior uterine cervical cancer, the patient was diagnosed with EBM from uterine cervical cancer. HPV genotyping may aid in discriminating EBM from primary bronchogenic carcinoma in patients with uterine cervical cancer.

Notch3 and Jagged2 contribute to gastric cancer development and to glandular differentiation associated with MUC2 and MUC5AC expression.

AIMS: Notch signalling plays diverse roles in malignant tumours as well as in normal tissue development. In this study we investigated the expression of Notch signalling pathway genes and their clinicopathological significance in gastric carcinomas. METHODS AND RESULTS: Notch1, Notch3, Jagged1, Jagged2 and Hes1 expression were analysed by quantitative real-time polymerase chain reaction (qRT-PCR) (n = 81) and immunohistochemistry (n = 103) in gastric carcinomas. MUC2 and MUC5AC expression were also assessed, using immunohistochemistry only. With qRT-PCR, Notch1, Notch3, Jagged1 and Jagged2 expression were increased significantly in tumour compared to normal tissue (P < 0.001, P = 0.002, P = 0.008 and P < 0.001, respectively). Overexpression of Notch3 and Jagged2 was associated with intestinal-type carcinomas (P = 0.024) and better histological differentiation (P = 0.047), respectively. Immunohistochemistry showed a reverse correlation between MUC2 and Notch3 or Jagged1 (P = 0.033 and P = 0.005, respectively) and between MUC5AC and Jagged1 or Hes1 (P = 0.004 and P = 0.002, respectively). Notch3 and Jagged2 gene overexpression related to a favourable outcome on univariate (P = 0.046 and P = 0.042, respectively) and multivariate (P = 0.045, Notch3) analysis. CONCLUSION: The expression of Notch3 and Jagged2 is associated not only with gastric cancer development but also with the intestinal/glandular differentiation of gastric carcinoma cells, suggesting a role as a possible favourable prognostic indicator.

Significance of cell cycle regulatory proteins as malignant and prognostic biomarkers in ovarian epithelial tumors.

The principal objective of this study was to comprehensively assess the clinicopathologic and prognostic impacts of the expression of various cell cycle regulatory proteins in patients with ovarian epithelial tumors. The tissue microarrays were constructed from formalin-fixed, paraffin-embedded tissues of 205 ovarian epithelial tumors. We investigated 55 benign (20 serous cystadenomas, 17 mucinous cystadenomas, and 18 endometriotic cysts), 72 borderline (26 serous borderline tumors and 46 mucinous borderline tumors), and 78 malignant tumors (45 serous carcinomas, 10 mucinous adenocarcinomas, 15 endometrioid adenocarcinomas, and 8 clear cell carcinomas). Immunohistochemical staining was performed using antibodies to p16(Ink4a), p53, p21(Waf1/Cip1), p27(Kip1), Cyclin D1, Cyclin E, Cyclin A, Cyclin B1, and Cyclin-dependent Kinase2. We noted significantly different levels of p53 and Cyclin B1 expressions between malignant and borderline tumors and between borderline and benign tumors excepting the mucinous type. The p21(Waf1/Cip1) and Cyclin A were significantly overexpressed in malignant tumors compared with borderline tumors. Cyclin A, Cyclin E, and p16(Ink4a) were more pronounced proteins, showing differential expression patterns among the histologic types of ovarian carcinomas. We determined that the overexpression of p16(Ink4a) (P=0.031), Cyclin E (P=0.009), and Cyclin-dependent Kinase2 (P=0.004) were significantly associated with higher tumor grades. Overexpression of p16(Ink4a) was correlated with both lymph node metastasis (P=0.030) and distant metastasis (P=0.034). Overexpression of Cyclin E was associated with advanced stage (P=0.004). Higher tumor grade (P=0.008), advanced stage (P=0.001), mucinous histologic type (P=0.012), low expression levels of p16(Ink4a) (P=0.032), and overexpression of p53 (P=0.032) were associated with poor overall survival on multivariate analysis in patients with ovarian cancer. In serous carcinomas, old age (P=0.005), distant metastasis (P=0.020), low expression of p16(Ink4a) (P=0.047), and overexpression of cytoplasmic p27(Kip1) (P=0.007) and Cyclin A (P=0.031) were all independent predictors of worse overall survival. Our data indicate that the overexpression of p16 and Cyclin E are valuable factors predicting disease progression and metastatic potential. Low p16(Ink4a) expression, overexpression of p53, cytoplasmic p27(Kip1), and Cyclin A are predictive markers for shorter overall survival in ovarian carcinomas.

p16Ink4a suppression of lung adenocarcinoma by Bmi-1 in the presence of p38 activation.

PURPOSE: Because evasion of tumor suppression is a critical step in cancer development, cancer cells have developed a variety of mechanisms to circumvent the influence of tumor suppressive pathways. Thus, genes that negatively regulate tumor suppressors could be considered novel types of oncogenes such as Bmi-1 repressing p16Ink4a and inhibiting p53 and were found to be frequently up-regulated in a variety of cancers. p38 mitogen-activated protein kinase (MAPK), which reportedly plays a crucial role as a tumor suppressor, is activated in number of lung adenocarcinomas, which is seemingly at odds with its role as a tumor suppressor. METHODS: We examined 10 lung adenocarcinomas and corresponding normal tissues and determined the expression levels of a variety of tumor suppressor proteins through real-time polymerase chain reaction and immunohistochemistry and measured p38 MAPK activity by immunoblotting or immunohistochemistry analysis. In the in vitro cellular model, p38 activation by H-Ras and consequent senescence induction was achieved through retro-viral gene transduction. Similarly, the suppression of p16Ink4a by Bmi-1 after the introduction of H-Ras was achieved through transient transfection with cationic liposome. RESULTS: We detected several lung adenocarcinomas that were positive for activated p38 MAPK but evidenced reduced levels of p16Ink4a expression. The suppression of p16Ink4a occurred in parallel with an increase in Bmi-1 and/or p16Ink4a promoter hypermethylation. Consistent with these observations, the H-Ras-stimulated induction of p16Ink4a was suppressed significantly through the coexpression of Bmi-1 in vitro. DISCUSSION: These results demonstrate that the suppression of p16Ink4a by either the induction of Bmi-1 or the hypermethylation of p16Ink4 may be an important step in avoiding tumor surveillance by p38 MAPK during the development of lung cancer.