Matched Adjacent Tissues Research Articles

Abstract C008: MUC16 expression in pancreatic ductal adenocarcinoma patient samples after neoadjuvant chemotherapy

Abstract Purpose Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with low five-year survival rates. Currently, surgical resection is the only potentially curative treatment available to patients. However, few patients are eligible for surgery without receiving prior therapy due to disease progression. Of those that receive upfront surgery, up to 60% develop recurrent disease within 5 years. Therefore, there is a need to increase the rate of complete surgical resections and the number of eligible patients for surgery. In the last decade, neoadjuvant therapy or treatment prior to surgery has increased for patients diagnosed with borderline resectable and locally advanced PDAC. Often, this therapy consists of chemotherapy alone or chemoradiotherapy treatment intended to downstage the tumor and reduce micrometastatic disease. Fluorescence-guided surgery (FGS) is a potential tool to increase the rate of complete surgical resections by specifically targeting a protein biomarker upregulated in the tumor with a fluorescent dye conjugated probe. Previous in vitro and in vivo research from our lab has indicated that the transmembrane mucin, MUC16, is a promising target for FGS in PDAC. However, protein expression has the potential to deviate after chemotherapy treatment. Therefore, this research aimed to determine the effect of neoadjuvant chemotherapy treatment (NCT) on the FGS-targetable biomarker, MUC16, in PDAC patient specimens. Experimental Procedures Deidentified PDAC and matched adjacent patient specimens receiving the NCT regimen or surgery-first treatment were obtained from UNMC Pathology and the UNMC Paraffin Tissue Bank. Deidentified normal pancreas specimens were received from the Normal Organ Recovery program, UNMC. MUC16 expression was investigated in each specimen. The presence of PDAC tumor and MUC16 stain expression patterns were confirmed by a specimen-blinded, board-certified pathologist. The expression of MUC16 between the NCT, surgery-first, and normal pancreas specimens was compared via statistical analysis. Results MUC16 was expressed in both the NCT and surgery-first specimens. There was a significant difference in MUC16 expression between the NCT specimens and both the normal pancreas (P = 0.0450) and surgery-first (P = 0.0494) specimens. In matched adjacent tissue, MUC16 expression was found to be higher in the NCT (P = 0.0003) and surgery-first (P = 0.1446) tumor specimens allowing for distinct tumor margins; however, this difference was not significant in the surgery-first specimens. Conclusions MUC16 expression retention in NCT PDAC specimens may indicate that MUC16 is a stable biomarker after chemotherapy treatment. Distinct MUC16 expression between tumor and matched adjacent specimens indicates the ability to achieve distinguishable tumor margins allowing for complete tumor resection. Therefore, MUC16 remains targetable for FGS in chemotherapy-treated patients. This has the potential to broaden the benefiting patient population and increase the number of patients that may undergo a complete surgical resection. Citation Format: Kathryn M. Muilenburg, Evie G. Ehrhorn, Madeline T. Olson, Carly C. Isder, Kelsey A. Klute, Geoffrey A. Talmon, Mark A. Carlson, Quan P. Ly, Aaron M. Mohs. MUC16 expression in pancreatic ductal adenocarcinoma patient samples after neoadjuvant chemotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C008.

Integrated bioinformatics analysis reveals correlations of high TRIM59 expression with worse prognosis and immune infiltrates in lung adenocarcinoma

Abstract Objective: Most patients with advanced lung cancer have a poor prognosis. Recent studies have identified TRIM59 as a novel molecule that serves as a prognostic factor for the progression of non-small cell lung cancer. In the present study, we investigated the role of TRIM59 in predicting the prognosis of lung adenocarcinoma (LUAD) as well as the correlation between TRIM59 expression and immune infiltrates. Methods: We analyzed TRIM59 expression in normal and tumor tissues based on RNA-sequencing datasets from The Cancer Genome Atlas and Genotype-Tissue Expression databases. Forty-seven cases of LUAD tissues and their matching adjacent tissues were collected, and TRIM59 expression in tissue samples was demonstrated by immunohistochemistry. All tissue specimens were obtained under the approval of the Medical Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine (approval No. IR2019001101; approved on April 3, 2019). The immune cell scores were calculated using the CIBERSORT database. The Tumor Immune Estimation Resource database was used to analyze the correlation between TRIM59 and immune cell activities. Results: TRIM59 was up-regulated in most cancer types. High TRIM59 expression predicted a worse prognosis in patients with LUAD (overall survival, P = 0.00096; disease-specific survival, P = 0.00056; disease-free interval, P = 0.0009; progression-free interval, P = 0.0012). Moreover, TRIM59 was highly expressed in patients with LUAD who had a poorer prognosis. TRIM59 also showed a significant correlation with the ESTIMATE score (P = 0.04) and stromal score (P = 0.005) in patients with LUAD. Notably, a significant correlation between TRIM59 and the tumor mutation burden was found in LUAD but in no other cancer types (P < 0.001). Further investigation showed that TRIM59 had a significant correlation with gene markers on neutrophils and dendritic cells. Conclusion: TRIM59 is a potential prognosticator in LUAD and may be correlated with immune cell identification, immune cell infiltration, and immunotherapy checkpoints in LUAD.

Assessment of the Diagnostic Value of GFAP, IGFBP-2, and YKL-40 in Patients with Glioblastoma

Background and Aims: An effective marker search in glioblastoma is precious in controlling and detecting the progression and monitoring of patients with glioblastoma. In this regard, the present study aimed to evaluate the diagnostic and prognostic role of glial fibrillary acidic protein (GFAP), insulin-like growth factor-binding protein -2 (IGFBP-2), and chitinase-3-like protein -1 (YKL-40) tissue and plasma levels in patients with GBM. Materials and Methods: A total of 22 patients with newly diagnosed glioblastoma (the fourth grade of glioma) who had undergone surgery at the Erfan Hospital were included in the current study. The levels of GFAP, IGFBP-2 were evaluated in 22 tumor tissues, and non-timorous matched adjacent tissue samples of patients with glioblastoma using the enzyme-linked immunosorbent assay . Besides, 22 healthy subjects with no history of glioblastoma served as controls for plasma samples. All analyses were evaluated using the SPSS version 22.0. Results: The tissue levels of GFAP, IGFBP-2, and YKL-40 were significantly higher in patients with glioblastoma when compared to the healthy controls (p=0.001). Nevertheless, there was no significant difference in comparison to the healthy control group in the plasma samples. Conclusions: Tissue levels of GFAP, IGFBP-2, and YKL-40 may be potential biomarkers for predicting and the progression in patients with glioblastoma.

Identification of key genes of papillary thyroid carcinoma by integrated bioinformatics analysis.

Background: Papillary thyroid carcinoma (PTC) is one of the fastest-growing malignant tumor types of thyroid cancer. Therefore, identifying the interaction of genes in PTC is crucial for elucidating its pathogenesis and finding more specific molecular biomarkers.Methods: Four pairs of PTC tissues and adjacent tissues were sequenced using RNA-Seq, and 3745 differentially expressed genes were screened (P<0.05, |logFC|>1). The enrichment analysis indicated that the vast majority of differentially expressed genes (DEGs) may play a positive role in the development of cancer. Then, the significant modules were analyzed using Cytoscape software in the protein–protein interaction network. Survival analysis, TNM analysis, and immune infiltration analysis of key genes were analyzed. And the expression of ADORA1, APOE, and LPAR5 genes were verified by qPCR in PTC compared with matching adjacent tissues.Results: Twenty-five genes were identified as hub genes with nodes greater than 10. The expression of 25 genes were verified by the GEPIA database, and the overall survival and disease-free survival analyses were conducted with Kaplan–Meier plotter. We found only three genes were confirmed with our validation and were statistically significant in PTC, namely ADORA1, APOE, and LPAR5. Further analysis found that the mRNA levels and methylation degree of these three genes were significantly correlated with the TNM staging of PTC. And these three genes were related to PTC immune infiltration. Verification of the expression of these three genes by RT-qPCR and Western blot further confirmed the reliability of our results.Conclusion: Our study identified three genes that may play key regulatory roles in the development, metastasis, and immune infiltration of papillary thyroid carcinoma.

The role and molecular mechanism of Trop2 induced epithelial-mesenchymal transition through mediated β-catenin in gastric cancer.

The present study elucidates the potential role of Trop2 in tumor invasion and the promotion of epithelial‐mesenchymal transition (EMT) when binding β‐catenin in GC. The role of Trop2 in promoting EMT in GC cells was examined by a variety of experimental assays. Moreover, the underlying molecular mechanism of Trop2 in promoting EMT was studied by in vivo and in vitro assays. The Trop2 expression in relation to tumor metastasis status was detected by IHC in 248 cases of GC tissues and 86 cases of matched adjacent tissues. Trop2 promoted the metastasis and induces EMT in GC. Meanwhile, the elevated protein levels of Trop2 and mesenchymal markers were also found in the TGF‐β1‐induced EMT model in GC cells. Importantly, Trop2 physically bound and activated β‐catenin to promote EMT; moreover, Trop2 increased the accumulation of β‐catenin in the nucleus to accelerate metastasis in GC cells. Inhibition of Trop2 expression in GC cells prevented the migration and invasion of GC cells in vivo. Trop2+/vimentin+ expression was higher in GC tissues than that in matched adjacent tissues, and Trop2+/vimentin+ expression in GC was associated with the differentiation, TNM stage, and distant metastases. These sets of data reveal a novel regulatory network of Trop2 in EMT and GC metastasis, suggesting Trop2 as a useful marker for inducing EMT and metastasis of GC, which may help to lead a better understanding of the pathogenesis of the GC.

Exosomal TRIM3 is a novel marker and therapy target for gastric cancer

BackgroundExosomes are critically involved in cancer development and progression. The exosomal contents have been suggested as ideal cancer biomarkers. In this study, we investigated the expression of exosomal proteins in the serum of gastric cancer patients and their roles in gastric cancer.MethodsThe proteomic profile of exosomes from the serum of gastric cancer patients was detected by using LC-MS/MS. The expression of TRIM3 in exosomes from the serum of gastric cancer patients and healthy controls was assessed by ELISA and western blot. Immunohistochemistry was used to detect TRIM3 expression in gastric cancer tissues and their matching adjacent tissues. The growth and migration abilities of gastric cancer cells with TRIM3 overexpression or knockdown in vitro were evaluated by colony formation assay and transwell migration assay. The effects of TRIM3 overexpression or knockdown on gastric cancer growth and metastasis in vivo were investigated by using subcutaneous xenograft tumor and peritoneal metastasis mouse model. The effects of TRIM3-overexpressing exosomes on gastric cancer growth and metastasis in vitro and in vivo were also evaluated.ResultsWe found that the expression levels of TRIM3 mRNA and protein were decreased in gastric cancer tissues compared to the matched control tissues. In addition, the levels of TRIM3 protein in the serum exosomes of gastric cancer patients were lower than that in healthy controls. We demonstrated that TRIM3 overexpression reduced while TRIM3 knockdown promoted the growth and metastasis of gastric cancer in vitro and in vivo through the regulation of stem cell factors and EMT regulators. Moreover, exosomes-mediated delivery of TRIM3 protein could suppress gastric cancer growth and metastasis in vitro and in vivo.ConclusionsTaken together, our findings suggest that exosomal TRIM3 may serve as a biomarker for gastric cancer diagnosis and the delivery of TRIM3 by exosomes may provide a new avenue for gastric cancer therapy.

KRAB-containing zinc finger protein ZNF496 inhibits breast cancer cell proliferation by selectively repressing ERα activity

KRAB-containing zinc finger proteins (KZNF) constitute the largest family of transcriptional regulators in humans and play critical roles in normal development and tumorigenesis. However, the function and mechanism of most KZNFs remain unclear. Here, we report that ZNF496, a KZNF family member, interacts with the DNA binding domain (DBD) of estrogen receptor alpha (ERα) via its C2H2 domain. This interaction decreases ERα binding to chromatin DNA and results in the repression of ERα transactivation, the selective suppression of ERα target genes, and ultimately in a reduction of ERα-positive cell growth in the presence of E2. An analysis of clinical data revealed that the downregulation of ZNF496 expression is observed only in ERα-positive and not in ERα-negative breast cancer tissues when compared with that in matched adjacent tissues. Lastly, we also observed that the downregulation of ZNF496 is associated with poor recurrence-free survival among patients with breast cancer. Collectively, our findings demonstrate that ZNF496 is a novel ERα-binding protein that acts as a target gene-specific ERα corepressor and inhibits the growth of ERα-positive breast cancer cells.

Abstract 4977: MIR1307 mediates pancreatic cancer resistance to FOLFIRINOX chemotherapy by affecting response to DNA damage

Abstract ntroduction: FOLFIRINOX-regime is a combination-chemotherapy that provides the best clinical benefit in pancreatic cancer (PC) patients, but is associated with severe toxicity. Aim of this study is to explore the role of miRNAs (MIR) as modulators of chemosensitivity and their potential as biomarkers of sensitivity to FOLFIRINOX-chemotherapy. Methods: High-throughput-screening (HTS) of 997 LNA-MIR-inhibitors was performed in PC cell lines (Capan1, MiaPaCa2) treated with a combination of Fluorouracil (F), Oxaliplatin (O) and Irinotecan (I) that mimics FOLFIRINOX-regime. Cell viability was monitored by CellTiter-Blue assay. Validation experiments were carried out with miRvana probes. MIR expression was assessed by TaqMan-assay. Apoptosis was measured by Flow-cytometry and western-blotting. Knock-out of microRNA was acheived by CRISPR-CAS9 in MiaPaca2 cells (MIR1307KO). Results: 41 and 84 miRNA-inhibitors enhanced FOI activity by &amp;gt;30% (p&amp;lt;0.001) in Capan1 and MiaPaCa2. These included MIR1307-inhibitor that was validated in Capan1, MiaPaCa2, Panc1, AspC1, BxPC3, and Su86.86 cell lines. The proportion of cells killed by FOI in comparison to DMSO was greater in cells transfected with MIR1307 inhibitor when compared to NEG CTRL, making the effect of this MIR specific for chemotherapy. MIR1307 was over-expressed in tumour compared to matched-adjacent tissue in 40/60 human PC cases, confirming clinical relevance. MIR1307KO cells were more sensitive to FOI than WT cells. Chemotherapy-induced apoptosis was higher in MIR1307KO cells (caspase 3/7 activity and annexin-V positivity). We observed significant upregulation of different markers of DNA damage (pH2AX2, 8OHdG, DNA breaks in Comet assay) in MIR1307KO cells treated with FOI in comparison to WT treated cells. Re-expression of MIR1307 in MIR1307KO cells could increase resistance to FOI chemotherapy and protected from FOI-induced DNA damage. Bioinformatics analysis identified MIR1307 binding-sites within a number of genes involved in the DNA-repair pathway (p&amp;lt;0.001, folding energy value greater than -12 Kcal/mol). Conclusions: We identified miR-1307 as a potential modulator of sensitivity to FOI-chemotherapy in PC. We showed that miR-1307 inhibition impairs the ability of PC cells to recover from chemotherapy damage and therefore enhances its activity. The assessemnt of it potential as predictive biomarker of response in PC patients is ongoing. Citation Format: Pietro Carotenuto, Domenico Zito, Maria C. Previdi, Maya Raj, Matteo Fassan, Andrea Lampis, Francesco Scalafani, Andrea Lanese, Ian Said-Huntingford, Jens C. Hahne, Kate Young, Ruwaida Begum, Zakaria Ethiar, Andrew Wotherspoon, Naureen Starling, Anguraj Sadanandam, David Cunningham, Ian Chau, Paul Workman, Rajesh Chopra, Nicola Valeri, Chiara Braconi. MIR1307 mediates pancreatic cancer resistance to FOLFIRINOX chemotherapy by affecting response to DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4977.

Suppressive effect of microRNA-138 on the proliferation and invasion of osteosarcoma cells via targeting SIRT1.

MicroRNAs (miRs), a class of small non-coding RNAs, function as key regulators in gene expression through binding to the 3′-untranslated region (UTR) of their target mRNA, which further leads to translational repression or mRNA degradation. Recently, miR-138 has been found to have a tumor suppressive role in a variety of human malignancies. However, the exact role of miR-138 in regulating the malignant phenotypes of osteosarcoma (OS) has remained to be elucidated. In the present study, reverse-transcription PCR analysis showed that the expression of miR-138 was markedly reduced in OS tissues compared to that in matched adjacent non-tumorous tissues. Furthermore, it was also downregulated in several common OS cell lines, when compared with that in a normal human osteoblast cell line. Overexpression of miR-138 suppressed cell proliferation and invasion and led to a significant decrease in the protein expression of sirtuin 1 (SIRT1), which was further identified as a direct target gene of miR-138 in MG63 cells. Moreover, restoration of SIRT1 expression reversed the suppressive effects of miR-138 on MG63 cell proliferation and invasion. Finally, the expression of SIRT1 was found to be significantly upregulated in OS tissues compared to that in matched adjacent tissues, and SIRT1 levels were inversely correlated with the miR-138 levels in OS tissues. Therefore, the present study demonstrated that miR-138 has a role in inhibiting OS cell proliferation and invasion via directly targeting SIRT1, and suggested that the miR-138/SIRT1 axis may become a promising therapeutic target for OS.

Analysis of gastric cancer tissues genome methylation by DNA methylation chip

To detect the methylation status of gastric cancer tissue genome by DNA methylation chip. Methylation status of 6 samples of gastric cancer tissues and their matched adjacent tissues was analyzed using methylated DNA immunoprecipitation(MeDIP) combined with NibleGen chip. Significantly different methylated genes in promoter region and CpG island between two tissues were searched. Functions of these significantly different methylated genes were analyzed by Gene Ontology and Pathway assays. In gene promoter regions, 113 significantly different methylated genes were identified in gastric cancer tissues, such as SHP1, FGF8 and CSF2RA, while 161 significantly different methylated genes were identified in their matched adjacent tissues, such as TNF, IGF2 and BMP7. In the CpG islands, 123 significantly different methylated genes were identified in gastric cancer tissues, such as WNT2B, JAK2 and TPT1, while 139 significantly different methylated genes were identified in their matched adjacent tissues, such as TNFRSF4, HOXC8 and NFYA. These genes located on different chromosomes. In gastric cancer tissues, the 1st and the 4th chromosomes had the most (both 11), the 18th and the 20th chromosomes had the least(both 1). In matched adjacent normal tissues, the 11th chromosome had the most (17), and no significantly different methylated gene was found on Y chromosome. These genes involved in many functions, such as protein phosphorylation, regulating cellular catabolism, ion transport, enzyme activity, transcriptional regulation, cell division, cell cycle regulation, and signal transduction. There are significant differences between gastric cancer tissues and their matched adjacent tissues in DNA methylation. DNA methylation genes locate on different chromosomes, and their number and distribution vary widely. These genes may be associated with many pathways in carcinogenesis.

Gut microbiome compositional and functional differences between tumor and non-tumor adjacent tissues from cohorts from the US and Spain

Colorectal cancer (CRC) is the third most common cancer in the world and the second leading cause of cancer deaths in the US and Spain. The molecular mechanisms involved in the etiology of CRC are not yet elucidated due in part to the complexity of the human gut microbiota. In this study, we compared the microbiome composition of 90 tumor and matching adjacent tissue (adjacent) from cohorts from the US and Spain by 16S rRNA amplicon sequencing in order to determine the impact of the geographic origin on the CRC microbiome. Data showed a significantly (P < 0.05) higher Phylogenetic Diversity (PD) for the US (PD Adjacent = 26.3 ± 5.3, PD Tumor = 23.3 ± 6.2) compared to the Spanish cohort (PD Adjacent = 18.9 ± 5.9, PD Tumor = 18.7 ± 6.6) while no significant differences in bacterial diversity were observed between tumor and adjacent tissues for individuals from the same country. Adjacent tissues from the Spanish cohort were enriched in Firmicutes (SP = 43.9% and US = 22.2%, P = 0.0001) and Actinobacteria (SP = 1.6% and US = 0.5%, P = 0.0018) compared to US adjacent tissues, while adjacent tissues from the US had significantly higher abundances of Fusobacteria (US = 8.1% and SP = 1.5%, P = 0.0023) and Sinergistetes (US = 0.3% and SP = 0.1%, P = 0.0097). Comparisons between tumor and adjacent tissues in each cohort identified the genus Eikenella significantly over represented in US tumors (T = 0.024% and A = 0%, P = 0.03), and the genera Fusobacterium (T = 10.4% and A = 1.5%, P = <0.0001), Bulleida (T = 0.36% and A = 0.09%, P = 0.02), Gemella (T = 1.46% and A = 0.19%, P = 0.03), Parvimonas (T = 3.14% and A = 0.86%, P = 0.03), Campylobacter (T = 0.15% and A = 0.008%, P = 0.047), and Streptococcus (T = 2.84% and A = 2.19%, P = 0.05) significantly over represented in Spanish tumors. Predicted metagenome functional content from 16S rRNA surveys showed that bacterial motility proteins and proteins involved in flagellar assembly were over represented in adjacent tissues of both cohorts, while pathways involved in fatty acid biosynthesis, the MAPK signaling pathway, and bacterial toxins were over represented in tumors. Our study suggests that microbiome compositional and functional dissimilarities by geographic location should be taken in consideration when approaching CRC therapeutic options.

Abstract A05: The control of nuclear ERK activity by the DUSP phosphatases in colorectal cancer

Abstract The RAS/RAF/MEK/ERK pathway is suspected to be strongly implicated in malignant transformation of colorectal epithelial cells since KRAS and BRAF genes are mutated in up to 60% of human colorectal cancers (CRCs). Despite intensive efforts made in the understanding of KRAS and BRAF functions in colorectal carcinogenesis, little is known about the oncogenic regulation of ERK activity in CRC cells, especially by the ERK-specific nuclear DUSP phosphatases. We have recently demonstrated in CRC cells that ERK1/2 activity is confined to the cytoplasm because of a pervanadate-dependent phosphatase activity. Accordingly, DUSP4 mRNA was found to be highly expressed, in a MEK-dependent manner, in CRC cells. Thus, DUSP4 and other nuclear DUSPs may function as part of a negative feedback mechanism in the control of the duration and magnitude of nuclear ERK activation during colorectal carcinogenesis. We thus speculate that the modification of nuclear ERK activity might strongly impact on the oncogenic properties of CRC. Methods: Expression of DUSPs was analyzed by PCR in CRC cells and qPCR in human colorectal tumors from different stages. To elucidate the role of nuclear DUSPs, RNA interference was used to specifically silence their expression in human HT29 CRC cell line, a cell line exhibiting BRafV600E mutation. Inducible expression of a DUSP4 inactivated mutant (DUSP4C284S) was done in a model of IEC-6 cells that express an inducible form of BRaf oncogene (BRafV600E:ER). Results: 1- DUSP2, DUSP4 and DUSP5 mRNAs were found to be highly expressed, in a MEK-dependent manner, in CRC cells. 2- DUSP4 and DUSP5 mRNA levels were markedly increased in colorectal tumors in comparison to healthy matched adjacent tissues. 3- Silencing of DUSP 2, 4 or 5 in HT29 cells increased ERK phosphorylation levels and markedly reduced colony formation in soft agar. 4- Furthermore, expression of DUSP4C284S in BRafV600E-expressing IEC-6 cells increased ERK phosphorylation into the nucleus and markedly reduced colony formation in soft agar. Conclusion: We are currently searching by which mechanisms sustained nuclear ERK activity reduces colorectal tumor cell growth. Therefore, targeting nuclear DUSP activity could become a new strategy to treat CRC. Citation Format: Sebastien Cagnol, Nathalie Rivard. The control of nuclear ERK activity by the DUSP phosphatases in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A05. doi: 10.1158/1557-3125.RASONC14-A05

Downregulation of PAX6 by shRNA Inhibits Proliferation and Cell Cycle Progression of Human Non-Small Cell Lung Cancer Cell Lines

BackgroundThe transcription factor PAX6 is primarily expressed in embryos. PAX6 is also expressed in several tumors and plays an oncogenic role. However, little is known about the role of PAX6 in lung cancer.MethodsThe function of PAX6 in lung cancer cells was evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell proliferation, anchorage-independent growth, and cell cycle arrest. The changes of cyclin D1, pRB, ERK1/2, p38 expression caused by PAX6 inhibition were detected using western-blotting. The PAX6 mRNA level in 52 pairs of tumors and corresponding matched adjacent normal tissues from non-small cell lung cancer patients and lung cancer cell lines was detected by real-time PCR.ResultsSuppression of PAX6 expression inhibited cell growth and colony formation in A549 and H1299 cells. The percentage of cells in G1-phase increased when PAX6 expression was inhibited. The cyclin D1 protein level, as well as the pRB phosphorylation level, decreased as a result of PAX6 down-regulation. The activity of ERK1/2 and p38 was also suppressed in PAX6 knock-down cells. The PAX6 mRNA was highly expressed in lung cancer tissue and lung cancer cell lines. In most patients (about 65%), the relative ratio of PAX6 mRNA in primary NSCLC versus adjacent tissues exceeded 100.ConclusionsOur data implicated that PAX6 accelerates cell cycle progression by activating MAPK signal pathway. PAX6 mRNA levels were significantly elevated in primary lung cancer tissues compared to their matched adjacent tissues.

Let-7 MicroRNA Transfer in Pancreatic Cancer-Derived Cells Inhibits In Vitro Cell Proliferation but Fails to Alter Tumor Progression

Pancreatic ductal adenocarcinoma (PDAC) is still the fourth leading cause of cancer-related deaths in Western countries, with increasing incidence. Neither effective prognostic markers nor therapies exist for this cancer. MicroRNAs are potent inhibitors of protein translation, and aberrantly expressed in many cancers. Because let-7 microRNA targets the K-ras oncogene, we aimed to characterize let-7 expression and function in PDAC in vitro and in vivo. Let-7 expression was quantified by real-time RT-PCR from resected tumors and matching adjacent tissue, and in endoscopic ultrasound-guided fine needle aspiration material from patients with PDAC. Let-7 is detected by reverse transcription in situ PCR in a PDAC tissue microarray. PDAC-derived cells were transfected with plasmid-based synthetic microRNAs or by lentiviral transduction, in vitro and in vivo. Let-7 microRNA expression is strongly reduced in PDAC samples, as compared with adjacent tissue. Let-7 is present in normal acinar pancreatic cells, and lost in poorly differentiated cancer samples. In addition, let-7 expression was repressed in patients with PDAC not eligible for surgery. Restoring let-7 levels in cancer-derived cell lines strongly inhibits cell proliferation, K-ras expression, and mitogen-activated protein kinase activation, but fails to impede tumor growth progression after intratumoral gene transfer or after implantation of Capan-1 cells stably overexpressing let-7 microRNA. We describe here for the first time the extensive loss of expression of let-7 in PDAC. In addition, this study provides the initial steps for a microRNA replacement therapy for this cancer.