Lymph Node Metastasis Specimens Research Articles

Abstract 1388: Spatial transcriptome analysis of EMT trait and corresponding immune composition study in HNSCC

Abstract Head and neck squamous cell carcinoma (HNSCC) is a prevalent malignant cancer worldwide. Conventional treatments for this type of cancer involve surgical resection, radiation therapy, and chemotherapy. However, in cases where the tumor recurs or metastasizes distantly, rendering surgical resection or radiation therapy impractical, the only viable option is the utilization of targeted drugs in conjunction with chemotherapy. Despite such interventions, the prognosis remains grim. Hence, the identification of genetic characteristics or biomarkers associated with easy metastasis or recurrence in the early stages of the tumor has become imperative for effective head and neck cancer treatment. The objective of this study is to analyze tumor specimens from the primary site and lymph node metastasis post-surgical resection in HNSCC patients. We employed the GeoMx Digital Spatial Profiler (DSP) for imaging-based digitized gene expression analysis. The DSP provides gene expression information at specific locations we selected, such as the primary tumor center core, primary tumor invasion borders, and lymph node metastasis specimens. The GeoMx DSP spatial analysis tool illustrates the cancer transcriptome atlas (CTA), encompassing the expression of 1825 genes related to cancer and immunity from the primary site to the metastatic site. We identified tissue-specific EMT gene expression patterns in paired tumor regions of interest (ROIs). ITGB1 emerged as the most significant co-expression marker in the primary outer tumor and metastatic tumor. Distinct EMT signature genes expressed in the HNSCC primary site and lymph node metastatic site were also discovered. Additionally, we conducted CTA profiling in correlated stroma ROIs. Through myeloid activity analysis in HNSCC paired samples, we identified distinct myeloid activation signatures in primary and metastatic site stroma cells. These results will provide crucial insights into head and neck cancer treatment strategies based on different tumor site characteristics. Citation Format: Chih-Hung Chung, Muh-Hwa Yang. Spatial transcriptome analysis of EMT trait and corresponding immune composition study in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1388.

Tumor expression of Nectin-1–4 and its clinical implication in muscle invasive bladder cancer: An intra-patient variability of Nectin-4 expression

There is limited evidence regarding the tumor expression of nectins and their clinical implications in muscle invasive bladder cancer. Herein, we evaluated expression of Nectins 1–4 in 64 patients with muscle invasive bladder cancer who underwent radical cystectomy using a histochemical scoring method (H-score; immunohistochemical staining intensity multiplied by the percentage of positive-staining cells). The cutoff values were defined based on the median H-scores. Of the 64 patients, 45 (70%) had residual tumors in radical cystectomy specimens, while 13 (20%) had lymph node metastasis. The median (interquartile range) H-scores of Nectin-1, − 2, − 3, and − 4 expression were 0 (0−10), 80 (30−180), 5 (0−30), and 100 (33−160), respectively. The Nectin-4 H-score of the neuroendocrine variant was significantly lower than that of pure urothelial carcinoma (P = 0.015). Post-neoadjuvant chemotherapy pathological response (<ypT2N0 residual disease and pN-negative) was achieved in 18 (49%) of the 37 patients who received neoadjuvant chemotherapy. Clinical stage II, not Nectin expression, was an independent factor associated with pathological response (P = 0.019, adjusted odds ratio 6.9, vs stage III/IV). There was no correlation in Nectin-4 tumor expression between transurethral resection and matched radical cystectomy specimens and between radical cystectomy specimens and matched lymph node metastatic lesions. However, there was a significant decrease in Nectin-4 expression in post-neoadjuvant chemotherapy radical cystectomy specimens compared to pre-neoadjuvant chemotherapy transurethral resection specimens (P = 0.008). Given the downregulation of Nectin-4 by chemotherapy and the significant discrepancy between radical cystectomy and matched lymph node metastasis specimens, baseline primary tumors may not be a suitable material for evaluating Nectin-4 expression and its potential as a predictive biomarker for enfortumab vedotin treatment.

Inactivation of homeodomain-interacting protein kinase 2 promotes oral squamous cell carcinoma metastasis through inhibition of P53-dependent E-cadherin expression.

Homeodomain‐interacting protein kinase 2 (HIPK2), a well‐known tumor suppressor, shows contradictory expression patterns in different cancers. This study was undertaken to clarify HIPK2 expression in oral squamous cell carcinoma (OSCC) and to reveal the potential mechanism of HIPK2 involvement in OSCC metastasis. Two hundred and four OSCC tissues, together with paired adjacent normal epithelia, dysplastic epithelia, and lymph node metastasis specimens, were collected to profile HIPK2 expression by immunohistochemical staining. High throughput RNA‐sequencing was used to detect the dysregulated signaling pathways in HIPK2‐deficient OSCC cells. Transwell assay and lymphatic metastatic orthotopic mouse model assay were undertaken to identify the effect of HIPK2 on tumor invasion. Western blotting and luciferase reporter assay were used to examine the HIPK2/P53/E‐cadherin axis in OSCC. Nuclear delocalization of HIPK2 was observed during oral epithelial cancerization progression and was associated with cervical lymph node metastasis and poor outcome. Depletion of HIPK2 promoted tumor cell invasion in vitro and facilitated cervical lymph node metastasis in vivo. According to mRNA‐sequencing, pathways closely related to tumor invasion were notably activated. Homeodomain‐interacting protein kinase 2 was found to trigger E‐cadherin expression by mediating P53, which directly targets the CDH1 (coding E‐cadherin) promoter. Restoring P53 expression rescued the E‐cadherin suppression induced by HIPK2 deficiency, whereas rescued cytoplasmic HIPK2 expression had no influence on the expression of E‐cadherin and cell mobility. Together, nuclear delocalization of HIPK2 might serve as a valuable negative biomarker for poor prognosis of OSCC and lymph node metastasis. The depletion of HIPK2 expression promoted OSCC metastasis by suppressing the P53/E‐cadherin axis, which might be a promising target for anticancer therapies.

LncRNA SLC26A4-AS1 suppresses the MRN complex-mediated DNA repair signaling and thyroid cancer metastasis by destabilizing DDX5.

Lymph node metastasis is the major adverse feature for recurrence and death of thyroid cancer patients. To identify lncRNAs involved in thyroid cancer metastasis, we systemically screened differentially expressed lncRNAs in lymph node metastasis, thyroid cancer, and normal tissues via RNAseq. We found that lncRNA SLC26A4-AS1 was continuously, significantly down-regulated in normal tissues, thyroid cancer, and lymph node metastasis specimens. Low SLC26A4-AS1 levels in tissues were significantly associated with poor prognosis of thyroid cancer patients. LncRNA SLC26A4-AS1 markedly inhibited migration, invasion, and metastasis capability of cancer cells in vitro and in vivo. Intriguingly, SLC26A4-AS1 could simultaneously interact with DDX5 and the E3 ligase TRIM25, which promoting DDX5 degradation through the ubiquitin-proteasome pathway. In particular, SLC26A4-AS1 inhibited expression of multiple DNA double-strand breaks (DSBs) repair genes, especially genes coding proteins in the MRE11/RAS50/NBS1 (MRN) complex. Enhanced interaction between DDX5 and transcriptional factor E2F1 due to silencing of SLC26A4-AS1 promoted binding of the DDX5-E2F1 complex at promoters of the MRN genes and, thus, stimulate the MRN/ATM dependent DSB signaling and thyroid cancer metastasis. Our study uncovered new insights into the biology driving thyroid cancer metastasis and highlights potentials of lncRNAs as future therapeutic targets again cancer metastasis.

Expression of fibroblast activation protein and the clinicopathological relevance in distal cholangiocarcinoma

Objectives: The current survival of patients with distal cholangiocarcinoma (dCCA) is poor. There is a need to develop new prognostic and predictive biomarkers to improve the survival of patients. Fibroblast activation protein (FAP) expression has been associated with survival in several solid malignancies. The goal of this study was to evaluate the expression pattern and prognostic significance of FAP in dCCA.Materials and methods: FAP expression was examined in 57 resected dCCA specimens and 28 paired lymph node metastasis specimens, as well as 10 benign bile ducts using immunohistochemistry. FAP expression was scored in the epithelial and stromal component of the dCCA specimens. The association between FAP expression and prognosis was evaluated using univariable and multivariable statistical modeling.Results: FAP expression was absent in the benign controls. FAP expression was evident in the epithelial 43 (75%) and stromal compartment 34 (60%) of dCCA. There was no association between epithelial or stromal FAP expression and clinicopathological factors. Epithelial FAP expression (HR 0.4 95% CI 0.20–0.78; p=.007) but not stromal FAP expression was significantly associated with better survival in univariable and multivariable analysis.Conclusions: FAP overexpression is evident in dCCA. There was a positive association between epithelial FAP expression and better survival which merits further evaluation.

Predictive value of phenotypic signatures of bladder cancer response to cisplatin-based neoadjuvant chemotherapy

BackgroundCisplatin-based neoadjuvant chemotherapy (NAC) for muscle-invasive urothelial carcinoma of the bladder confers only a modest survival advantage. Patients with pathologic progression on NAC have poor outcomes related to a delay in definitive surgical management. ObjectiveTo characterize the value of epithelial-mesenchymal transition (EMT) effectors and other novel biomarkers to predict response to NAC. MethodsA tissue microarray was constructed from patients with clinical stage T2 urothelial carcinoma of the bladder using transurethral resection (TUR) specimens (N = 69) and case-matched post-NAC cystectomy specimens (N = 51). Patients were stratified based on pathologic response to NAC and cancer-specific survival. Biomarker expression in TUR specimens was correlated with pathologic response to NAC and clinical outcomes. Phenotypic changes in expression induced by cisplatin-based NAC were characterized in primary TUR and post-NAC cystectomy and lymph node metastasis specimens. ResultsIncreased expression of mesenchymal markers and actin-cytoskeleton regulators, as well as low apoptosis index, in TUR specimens was associated with pathologic progression on cisplatin-based NAC. Overexpression of N-cadherin and decreased apoptosis was predictive of disease-specific mortality. NAC decreased cofilin phosphorylation and induced a mesenchymal-epithelial transition phenotype. ConclusionsThe epithelial-mesenchymal transition phenotype and actin-cytoskeleton remodeling are associated with pathologic progression on NAC. Chemotherapy induced an mesenchymal-epithelial transition phenotype and decreased cofilin phosphorylation, providing new insights into therapeutic resistance mechanisms. Primary tumors with high apoptosis rates exhibited favorable response to NAC and lower mortality rates, indicating that these tumors may be sensitized to therapy. Further validation will establish these novel signatures as predictors of therapeutic response.

Molecular Testing in EBUS-TBNA Specimens of Lung Adenocarcinoma: A Study of Concordance Between Cell Block Method and Liquid-Based Cytology in Appraising Sample Cellularity and EGFR Mutations.

Cytological endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) specimens of mediastinal lymph node metastasis are frequently used to perform concomitant diagnosis, staging and genetic testing in non-small-cell lung cancer (NSCLC). The purposes of this single-center retrospective study were to evaluate EBUS-TBNA samples' adequacy for molecular testingof epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK), and to analyze the concordance between the cell block method and liquid-based cytology (LBC) in appraising the sample cellularity and in detecting EGFR mutation. We retrospectively examined 82 patients who underwent EBUS-TBNA from October 2012 to September 2015 and received a confirmed diagnosis of lymph node metastasis of lung adenocarcinoma. Each sample was processed using both cell block and LBC to carry out DNA analysis (adequacy criterion: tumor cell percentage >25%) and EGFR mutation testing. Fifty-four patients were male, 66 were current or former-smokers, and the median age was 67 years. The median size of sampled lymph nodes was 14.8 mm. Seventy-one and 66 samples were adequate to perform cell block and LBC, respectively. The κ-statistic (0.78) showed an excellent concordance. EGFR mutation was detected in eight patients using cell block and in seven using LBC, with a simple percentage agreement of 87.5%. ALK translocation was found in two patients. This study demonstrates the feasibility of EGFR mutation analysis with both cell block and LBC, with an excellent concordance between the two methods. Considering that the majority of advanced NSCLCs are diagnosed on cytology specimens, LBC is feasible and needs to be implemented for ancillary tests (immunocytochemistry, molecular analysis).

Clinical Usefulness of the Histoculture Drug Response Assay for Breast Cancer.

The histoculture drug response assay (HDRA) was used to compare drug sensitivity of recurrent and primary breast cancer in vitro as well as in the clinic. The HDRA utilizes 3-dimensional culture of human tumors on Gelfoam®. The evaluation rate was 98.8%. The HDRA mean inhibition rate of primary tumors vs. recurrent tumors was, respectively,57.9% and 38.6% for doxorubicin (DOX); 59.9% and 42.8% for mitomycin C (MMC); 49.0% and 33.4% for 5-fluorouracil (5-FU); and 34.5% and 16.0% for cisplatinum (CDDP). The recurrent cases were pretreated clinically with CAF (cyclophosphamide [CTX], DOX, and 5-FU), CEF (CTX, epirubicin[EPN], and 5-FU), or CMF (CTX, methotrexate[MTX], and 5-FU). 64.7% of the recurrent cases were resistant to all four agents tested compared to 27% of the primary cases. Only 5.9% of the recurrent cases were sensitive to three or more agents as opposed to 18% of the primary cases. The correlation of the HDRA results to clinical outcome in another breast-cancer study was 80.0% with 15 cases evaluated consisting of five true positives, three false positives, seven true negatives, and no false negatives. HDRA was also performed on surgical specimens of primary tumor and axillary lymph node metastasis from each of 30 breast cancer patients. The lymph-node metastases were more resistant than the primary tumor for DOX, 5-FU, and MMC, but not for CDDP. The data suggest that both primary tumor and metastases from individual patients should be tested in the HDRA to enhance clinical efficacy of chemotherapy. There also was a lack of correlation with breast cancer subtype and drug response in the HDRA, further suggesting the importance of individualized treatment for breast cancer patients afforded by the HDRA.

TIMP1 is a prognostic marker for the progression and metastasis of colon cancer through FAK-PI3K/AKT and MAPK pathway.

BackgroundTissue inhibitor matrix metalloproteinase 1 (TIMP1) plays a vital role in carcinogenesis, yet its precise functional roles and regulation remain unclear. In this study, we aim to investigate its biological function and clinical significance in human colon cancer.MethodsWe analyzed the expression of TIMP1 in both public database (Oncomine and TCGA) and 94 cases of primary colon cancer and matched normal colon tissue specimens. The underlying mechanisms of altered TIMP1 expression on cell tumorigenesis, proliferation, and metastasis were explored in vitro and in vivo.ResultsTIMP1 was overexpressed in colon tumorous tissues and lymph node metastasis specimens than in normal tissues. The aberrant expression of TIMP1 was significantly associated with the regional lymph node metastasis (p = 0.033), distant metastasis (p = 0.039), vascular invasion (p = 0.024) and the American Joint Committee on Cancer (AJCC) stage (p = 0.026). Cox proportional hazards model showed that TIMP1 was an independent prognostic indicator of disease-free survival (HR = 2.603, 95 % CI: 1.115–6.077, p = 0.027) and overall survival (HR = 2.907, 95 % CI: 1.254–6.737, p = 0.013) for patients with colon cancer. Consistent with this, our findings highlight that suppression of TIMP1 expression decreased proliferation, and metastasis but increased apoptosis by inducing TIMP1 specific regulated FAK-PI3K/AKT and MAPK pathway.ConclusionTIMP1 might play an important role in promoting tumorigenesis and metastasis of human colon cancer and function as a potential prognostic indicator for colon cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0427-7) contains supplementary material, which is available to authorized users.

Karyopherin alpha 2 is a novel prognostic marker and a potential therapeutic target for colon cancer.

BackgroundKaryopherin alpha 2 (KPNA2), a member of the karyopherin family, plays a vital role in carcinogenesis. Yet its role in colon cancer is poorly characterized. We sought to clarify the clinical significance of its dysregulated expression in human colon tumor specimens.MethodsWe evaluated KPNA2 mRNA and protein expression by real-time polymerase chain reaction and Western blotting in 40 primary colon cancer tissues and paired adjacent normal colon mucosa specimens. KPNA2 protein expression in colon tissue microarray of tumor and normal tissue specimens and lymph node metastasis specimens obtained from 195 colon cancer patients were analyzed immunohistochemically. The effect of KPNA2 knockdown on carcinogenesis potential of human colon cancer cells was determined using Cell Counting Kit-8 (CCK8), colony formation, cell migration, and tumorigenesis in nude mice.ResultsKPNA2 was expressed at higher levels in colon tumors and lymph node metastasis specimens than in normal tissues. Patients with KPNA2-positive tumors were significantly correlated with the American Joint Committee on Cancer (AJCC) stage (p = 0.01), T-classification (p = 0.018), regional lymph node metastasis (p = 0.025), distant metastasis (p = 0.014), and differentiated degree (p = 0.001). KPNA2 was shown to be an independent prognostic indicator of disease-free survival (HR 1.681; 95 % CI: 1.170–2.416; p = 0.005) and overall survival (HR 2.770; 95 % CI: 1.314–5.837; p = 0.007) for patients with colon cancer. Knockdown of KPNA2 expression inhibited colon cancer cell proliferation, colony formation, and migration.ConclusionKPNA2 might play an important role in colorectal carcinogenesis and functions as a novel prognostic indicator and a potential therapeutic target for colorectal cancer.

The reduction of the mitochondrial cytochrome B makes development of gastric cancerthrough mitogen-activated protein kinase pathway

Objective Collect clinical specimens of gastric cancer patients with metastasis and without metastasis, application of molecular biology method confirm cytochrome B in its role in the development of cancer of the stomach, provide experimental basis for exploring the mechanism of gastric cancer. Methods Collect 12 cases confirmed by pathology of gastric cancer surgery in patients with gastric cancer tissue, including specimens with and without lymph node metastasis specimens each six cases. Detect the expression of two groups of gastric cancer tissue apoptosis related factors B cell lymphoma/leukemia- 2 associated X protein(bax), B cell lymphoma/leukemia- 2(bcl- 2), Caspase and mitogen- activated protein kinase(MAPK)signaling pathway proteins level by Western blotting. Results Compared with the group without metastasis, the expression of promoting apoptosis factor bax was reduced in gastric cancer tissue(0.55:1.00, P< 0.05), and the expression of inhibiting apoptosis factor bcl-2 increased(1.32:1.00, P<0.05), and the level of cell apoptosis decreased(2.86:3.35, P< 0.05). MAPK pathway activation level in gastric cancer tissue with metastasis was significantly reduced by comparing with the gastric cancer tissue without metastasis(P< 0.05). Conclusion Cytochrone B expression level decrease, thus inhibit MAPK pathway phosphorylation and reduce the cell apoptosis. Gastric cancer metastasis is related to the above mechanism. Key words: Gastric cancer; Mitochondrial cytochrome B; Apoptosis; Mitogen-activated protein kinase pathway

Epigenetic variations in breast cancer progression to lymph node metastasis.

Breast cancer is a heterogeneous disease characterized by the accumulation of genetic and epigenetic alterations that contribute to the development of regional and distant metastases. Lymph node metastasis (LNM) status is the single most important prognostic factor. Metastatic cancer cells share common molecular alterations with those of the primary tumor, but in addition, they develop distinct changes that allow the cancer to progress. There is an urgent need for molecular studies which focus on identifying genomic and epigenomic markers that can predict the progression to metastasis. The objective of this study was to identify epigenetic similarities and differences between paired primary breast tumor (PBT) and LNM. We employed Methylation-Specific-MLPA (Multiplex ligation-dependent probe amplification) to assess the methylation status of 33 cancer-related genes in a cohort of 50 paired PBT and LNM specimens. We found that the methylation index, which represents the degree of aberrantly methylated genes in a specimen, was maintained during the progression to LNM. However, some genes presented differential methylation profiles. Interestingly, PAX6 presented a significant negative correlation between paired PBT and LNM (p = 0.03), which indicated a switch from methylated to unmethylated status in the progression from PBT to LNM. We further identified that the methylation status of PAX6 on the identified CpG site functionally affected the expression of PAX6 at the mRNA level. Our study unraveled significant epigenetic changes during the progression from PBT to LNM, which may contribute to improved prognosis, prediction and therapeutic management of metastatic breast cancer patients.

Alterations in hormonal receptor expression and HER2 status between primary breast tumors and paired nodal metastases: discordance rates and prognosis.

We aimed to evaluate the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression discordance in matched pairs of primary breast cancer and lymph node metastasis specimens and determine the effect of discordance on prognosis. Among all patients diagnosed with lymph node metastases from 2004 to 2007, primary tumors and paired lymph node metastases were resected from 209 patients. The status of ER, PR, and HER2 expression was analyzed immunohistochemically in 200, 194, and 193 patients, respectively. Discordance was correlated with prognosis. Biomarker discordance between primary tumors and paired lymph node metastases was 25.0% (50/200) for ER status, 28.9% (56/194) for PR status, and 14.0% (27/193) for HER2 status. ER positivity was a significant independent predictor of improved survival when analyzed in primary tumors and lymph node metastases. Patients with PR-positive primary tumors and paired lymph node metastases displayed significantly enhanced survival compared to patients with PR-positive primary tumors and PR-negative lymph node metastases. Patients with ER- and PR-positive primary tumors and paired lymph node metastases who received endocrine therapy after surgery displayed significantly better survival than those not receiving endocrine therapy. Similalry treated patients with PR-negative primary tumors and PR-positive paired lymph node metastases also displayed better survival than those not receiving endocrine therapy. Biomarker discordance was observed in matched pairs of primary tumors and lymph node metastases. Such cases displayed poor survival. Thus, it is important to reassess receptor biomarkers used for lymph node metastases.

Abstract 5320: Sonic hedgehog in non-small cell lung cancer lymph node metastasis; ROS and PI3kinase/Akt signaling pathway

Abstract Sonic hedgehog signaling pathway plays an important role in the metastasis of various organs. Recently, we found that increased shh producing cells correlate with enhanced migration, invasiveness and increased lymphangiogenesis in Non-small-cell lung cancer (NSCLC), but the mechanism is unknown. In this study, we investigated the function of shh pathway in human NSCLC. We performed immunohistochemistry on lung tumor biopsies and the result showed the expressions of shh, Gli1, VEGF-D and LYVE1 were enhanced in lymph node metastasis specimens compared to lymph node negative lung cancer specimens. We also investigated the role of shh signaling in the invasiveness and the motility of NSCLC. We found that Shh N-terminal peptide (N-shh) enhanced cell motility and invasiveness in NSCLC, and this process was inhibited by AAD-cyclopamine (SMO inhibitor). We observed that the mRNA expressions of shh, Gli1, VEGF-D and LYVE1 were enhanced by N-shh treatment, whereas no increase of these factors was observed when KAAD-Cyclopamine (a Shh signaling inhibitor) was treated together. In addition, we confirmed that Shh-induced lymphangiogenesis and motility/invasiveness of NSCLC are also reduced by LY294002, PI-3 Kinase/Akt inhibitor meaning that shh pathway and PI-3 Kinase/Akt are strongly related. Thus, we investigated the functional relationship between Shh and Akt signaling in the metastatic progression in NSCLC. Phosphorylation of Akt was enhanced by N-Shh, but regulated by KAAD-cyclopamine or LY294002. We further analyzed the effects of shh signaling in vitro by stimulating a HMVEC with N-shh and we observed N-shh enhanced the tube formation on matrigel, but the enhancement was inhibited upon cyclopamine or LY294002 treatment. The ROS production was increased in shh-overexpressing cells. These results indicate that shh signaling pathway can contribute to ROS generation enhancing the metastatic and lymphangiogenetic potential of tumor cells. Our findings suggest that Shh-Akt signaling processes through ROS and the activation of these pathway increases the metastatic potential of non-small cell lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5320. doi:1538-7445.AM2012-5320

Runx3 expression in lymph nodes with metastasis is associated with the outcome of gastric cancer patients.

Accumulating evidence shows that runt-related transcription factor 3 (Runx3) is a putative tumor suppressor in various types of cancer, the lower levels of which are associated with a less favorable cancer outcome. However, these studies were restricted to primary cancer lesions. Lymph node metastasis (LNM) is a significant factor in determining the prognosis of patients with gastric cancer and is a frequent target of chemotherapy. In the present study, we investigated the expression of Runx3 in the lymph nodes (LNs) of stomach carcinoma and the association of Runx3 expression with the prognosis of patients. The expression of Runx3 in LNs with and without metastasis was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The positive rate of Runx3 mRNA in LNM specimens was significantly lower (28.4%, 21 out of 74) compared to that of the non-metastatic samples (33.3%, 9 out of 27, P<0.05). Similar findings were obtained by Western blotting. Univariate analysis revealed that the loss of Runx3 expression in LNs was not only associated with poor clinicopathological factors, such as LNM, distant organ metastasis, later clinicopathological stages and deep infiltration, but also with a lower 5-year survival rate and poorer prognosis. These results strongly suggest a potential diagnostic value of Runx3 expression in LNs and multiple pathways contributing to the outcome of patients with gastric cancer.

KLF4-Mediated Negative Regulation of IFITM3 Expression Plays a Critical Role in Colon Cancer Pathogenesis

IFITM3, an IFN-inducible gene, is overexpressed in human colorectal cancer. In this study, we sought to determine the clinical significance and underlying mechanisms of its dysregulated expression in human colon tumor specimens and murine models of this disease. IFITM3 expression in a tissue microarray of tumor and matched normal colon tissue specimens and lymph node metastasis specimens obtained from 203 patients with colon cancer was measured immunohistochemically. IFITM3 was expressed at higher levels in colon tumors and, particularly, nodal metastases than in normal colon tissue. A Cox proportional hazards model showed that IFITM3 expression was an independent prognostic factor for disease-free survival in patients with colon cancer. Knockdown of IFITM3 expression by a specific siRNA significantly suppressed the proliferation, colony formation, migration, and invasion of colon cancer cells in vitro and tumor growth and metastasis in a xenograft model. Restored expression of KLF4, a putative tumor suppressor, downregulated IFITM3 expression in colon cancer cells in vitro. Two KLF4-binding sites in the IFITM3 promoter bound specifically to KLF4 protein in a chromatin immunoprecipitation assay and promoter mutagenesis analyses. Specific deletion of KLF4 led to IFITM3 overexpression in colon mucosa in Villin-Cre(+);Klf4(fl/fl) mice. An inverse correlation between loss of KLF4 expression and IFITM3 overexpression was evident in human colon tumors. These clinical and mechanistic findings indicate that IFITM3 is a direct transcriptional target of KLF4 and that dysregulated KLF4 expression leads to aberrant IFITM3 expression, thus contributing to colon cancer progression and metastasis.

Abstract 1480: Reactive oxygen generated by shh signaling contributes to lymphangiogenesis in the non-small cell lung cancer

Abstract Approximately 90% of all cancer deaths arise from the metastatic spread of primary tumours. Recent reports have demonstrated that the Sonic hedgehog (shh) signaling pathway plays an important role in the metastasis of various organs, including the prostate, pancreas, gastrointestinal tract, esophageal and other tumors. Recently, we found that increased shh producing cells correlate with high migration, invasiveness and increased lymphangiogenesis in Non-small-cell lung cancer (NSCLC), but the mechanism is unknown. Furthermore, blockade of shh pathway by target antibodies inhibited shh-induced tumor growth, metastasis and lymphangiogenesis. In this study, we investigated the hypothesis that expression and function of the shh pathway in human Non-small cell lung cancer. We performed immunohistochemistry on lung tumor biopsies showed that expressions of shh, Gli-1, VEGF-D and LYVE-1 were enhanced in lymph node metastasis specimens compared with lymph node negative lung cancer specimens. To investigate the role of shh signaling as it relates to invasive phenotypes and the motility of NSCLC. We found that recombinant human shh (N-shh) enhanced cell motility and invasiveness in gastric cancer cells, whereas no increase was observed in cells that were treated with KAAD-cyclopamine(SMO inhibitor). The overall cell growth was not affected by treatment with N-Shh when cells were treated with the same concentration and for the same length of time, which indicates that the difference in motility was not due to an effect on proliferation. Reverse transcription-PCR analysis was performed to find lymphangiogenesis factor of mRNA levels. The expressions of shh, Gli-1, VEGF-D and LYVE-1 was also enhanced by treatment with N-shh, but not KAAD-cyclopamine. We further analysed the affects of shh signaling in vitro by stimulating a normal human lymphatic endothelial cell line (HMVEC) with N-shh. In vitro lymphangiogenesis assay using the HMVEC cell line. Stimulation with N-shh enhanced tube formation on matrigel. Stimulation with shh significantly increased in vitro lymphangiogenesis, but not KAAD-cyclopamine. To investigate the role and mechanism for shh signaling in regulating the lymphangiogenetic progression of NSCLC. Tumour hypoxia not only induces tumour angiogenesis, but also modulates the expression of several genes that have been implicated in tumour metastasis. We next explored how the shh signaling and resultant expression of LYVE-1 and VEGF-D regulate metastasis. Because expression of LYVE-1 and VEGF-D may lead to overproduction of reactive oxygen species (ROS), we estimated the amounts of ROS, and found that the N-shh in NSCLC showed enhanced ROS production, whereas the KAAD-cyclopamine did not. These results indicate that shh signaling pathway can contribute to ROS gerenates by enhancing the metastatic and lymphangiogenetic potential of tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1480. doi:10.1158/1538-7445.AM2011-1480

Four-protein signature accurately predicts lymph node metastasis and survival in oral squamous cell carcinoma

Abstract The presence of lymph node metastasis significantly affects the survival of oral squamous cell carcinoma patients and successful detection and removal of positive lymph nodes is crucial in the treatment of this disease. Current evaluation methods still have their limitations in detecting the presence of tumor cells in the lymph nodes, where up to a third of clinically diagnosed metastasis-negative (N0) patients actually have metastasis—positive LNs in the neck. We aimed to develop a molecular signature in the primary tumor that could predict lymph node (LN) metastasis in oral squamous cell carcinoma. The expression levels of 11 proteins was evaluated using immunohistochemical analysis in a tissue microarray (TMA) consisting of 110 specimens from 32 individuals. We used receiver operating characteristic (ROC) curve to identify proteins that could significantly differentiate patients with LN metastasis from those that did not. Unsupervised hierarchical clustering analysis was used to verify the ability of chosen biomarkers in segregating LN positive patients from those with no LN involvement. Kaplan-Meier survival curve and log rank test was utilized to determine the association between LN metastasis and biomarker expression with disease-specific survival. Of the 11 biomarkers, EGFR, HER-2/neu, LAMC2 and RHOC were found to be significantly associated with LN metastasis. Unsupervised hierarchical clustering demonstrated that expression patterns of these 4 proteins could be used to differentiate the positive LN metastasis specimens from specimens that are negative. Collectively, EGFR, HER-2/neu, LAMC2 and RHOC have a specificity of 87.5% and sensitivity of 70% with a prognostic accuracy of 83.4% for LN metastasis. We also demonstrated the LN signature could independently predict disease-specific survival (p = 0.023). In summary, we developed a 4-protein LN signature that could reliably distinguish patients with lymph node metastasis from those who were metastasis free. In addition, this LN signature is also associated with disease-specific survival indicating that would be a useful prognostic tool for the management of oral cancer patients.

Abstract 5195: Shh/Gli signaling pathway promotes tumor metastasis and lymphangiogenesis via activation of PI3K/Akt and Erk pathway in gastric cancer

Abstract The activation of Sonic hedgehog (Shh)/Gli signaling is involved in the progression and invasion of various tumors types, including gastric carcinoma. Epithelial-mesenchymal transition (EMT) and matrix metalloproteinases (MMPs) have been implicated in facilitating the invasion and metastatsis. Herein, we investigated the impact of phosphoinositide 3-kinase (PI3K)/Akt and Erk pathway and MMPs activity on the Shh/Gli-mediated EMT and invasion of gastric cancer cells. We found that overexpression of Shh significantly enhanced cellular motility/invasiveness, lymphangiogenesis, and metastatic ability of tumor cells in gastric cancer cell lines and mice. Stimulation of Shh in gastric cancer cells also induced a full EMT process (characterized by Snail induction and E-cadherin down-regulation) and expression and activity of MMP-9. Meanwhile, blockade of Shh/Gli signaling by Cyclopamine (a Shh signaling inhibitor), anti-Shh neutralizing antibodies, or Gli siRNA inhibited Shh-induced motility/invasiveness and lymphangiogenesis in gastric cancer by subsequently restoring these changes of EMT markers and activity of MMP-9. The phosphorylation of Akt and Erk was also enhanced by treatment with Shh, but not cyclopamine, anti-Shh neutralizing antibodies, or Gli siRNA. Blockade of the Akt and MEK kinase using kinase-dead Akt, LY294002, or PD98059 significantly inhibited the Shh-induced EMT, activity of MMP-9, lymphangiogenesis, and metastasis in tumor cell lines and mice over-expressing Shh. Furthermore, knock-down of MMP-9 by its siRNA results in an decrease in invasiveness of gastric cancer cells treatment with Shh. Immunohistochemistry on gastric tumor biopsies showed that the levels of Gli, E-cadherin, MMP-9, D2-40 (lymphangiogenic factor), and phospho-Akt expression were enhanced in cases of metastatic gastric cancer than in cases of primary gastric cancer. Moreover, the strong correlation between Gli and E-cadherin, MMP-9, D2-40, phospho-Akt, or phospho-Erk expression was also observed in lymph node metastasis specimens. These data indicate that Shh/Gli signaling pathway promotes tumor metastasis in gastric cancer through activation of PI3K/Akt and Erk pathway followed by up-regulation of EMT, MMP-9, and lymphangiogenesis, which results in the stimulation of invasiveness of tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5195.

Up-regulation of PIK3CA promotes metastasis in gastric carcinoma

To explore expressions of PIK3CA in the progression of gastric cancer from primary to metastasis and its effects on activation of phosphatidylinositol 3-kinase (PI3K)/Akt pathway. mRNA and protein levels of PIK3CA were assessed, respectively, by real-time quantitative polymerase chain reaction and immunohistochemistry in specimens of normal gastric mucosa, primary foci and lymph node and distant metastasis of gastric cancer. Akt and phosphorylated Akt protein were also examined by Western blotting in these tissues, in order to analyze the effect of PIK3CA expression level changes on the activation of PI3K/Akt signaling pathway. PIK3CA mRNA in lymph node metastasis were approximately 5 and 2 folds higher, respectively, than that in the corresponding normal gastric mucosa and primary gastric cancer tissues (P < 0.05), while no statistical significance was found compared with distant metastasis. Immunohistochemically, PIK3CA protein expression was discovered in 7 (35%) specimens of 20 primary foci vs 10 (67%) of 15 of lymph node metastasis or 11 (61%) of 18 of distant metastasis (35% vs 67%, P = 0.015; 35% vs 61%, P = 0.044). With the increased level of PIK3CA expression, the total Akt protein expression remained almost unchanged, but p-Akt protein was upregulated markedly. Increased expression of PIK3CA is expected to be a promising indicator of metastasis in gastric cancer. Up-regulation of PIK3CA may promote the metastasis of gastric cancer through aberrant activation of PI3K/Akt signaling.