Shorter Disease-free Survival Time Research Articles

THU550 The Splicing Factor SRSF6 Regulates Androgen Receptor Activity And Represents A Potential Therapeutic Target In Prostate Cancer

Abstract Disclosure: R.M. Luque: None. A.J. Montero-Hidalgo: None. E. Gomez-Gomez: None. D. Olmos: None. M.D. Gahete: None. J.M. Jimenez-Vacas: None. Background: Prostate cancer (PCa) is the fifth leading cause of cancer-related death worldwide. Finding novel therapeutic strategies to tackle PCa, especially its most advanced phenotype [i.e., castration-resistant prostate cancer (CRPC)], is urgently needed. In this sense, although the dysregulation of the splicing process has emerged as a distinctive feature of advanced PCa, the potential role that splicing regulators may play in this disease remain poorly understood. In this study, we aimed to analyse the levels, pathophysiological role and associated molecular landscape of the splicing factor SRSF6 in PCa. Methods: SRSF6 levels (mRNA, protein and CNA) were interrogated in seven well-characterized cohorts of PCa patients and in the Hi-MYC transgenic model. The effect of SRSF6 overexpression and silencing was tested in vitro (proliferation, migration, colony- and tumorspheres-formation) and in vivo (tumor growth of xenograft tumours). RNAseq was performed in 22Rv1 cells to analyse splicing and gene expression changes in response to SRSF6 silencing. Findings: Our results showed that SRSF6 levels (mRNA/protein) are upregulated in PCa vs non-tumour prostate samples, linked to clinical parameters of tumour-aggressiveness (e.g., Gleason score, T-stage, perineural infiltration, metastasis at diagnosis), and associated with poor prognosis (i.e., shorter disease-free survival time) in PCa patients. Moreover, SRSF6-overexpression increased, while its silencing decreased, functional parameters of aggressiveness in vitro and tumour growth in vivo. Mechanistically, SRSF6 modulation resulted in dysregulation of Androgen-Receptor (AR) activity through transcriptional control of APPBP2 and TOP2B. Discussion: SRSF6 could represent a new therapeutic target to inhibit persistent AR-signalling in advanced PCa. Funding: Spanish Ministry of Science, Innovation, and Universities (PID2019-105564RB-I00, FPU18/02485), Health Institute Carlos III (DTS20-00050), and CIBERobn. Presentation: Thursday, June 15, 2023

Clinical impact of body composition on postoperative outcomes during neoadjuvant chemoradiation therapy for distal bile duct cancer.

Body composition changes during neoadjuvant therapy and their clinical significance have not been clarified. The present study aimed to investigate body composition changes during neoadjuvant chemoradiation therapy (NACRT) in patients with distal bile duct cancer and the clinical impact on postoperative complications and the prognosis. A total of 16 patients with distal bile duct cancer who underwent curative resection after NACRT were retrospectively evaluated. The area of skeletal muscle, visceral fat and subcutaneous fat on computed tomography and immunological and nutritional indices were assessed before and after NACRT. All 16 patients completed NACRT followed by pancreaticoduodenectomy without mortality. There was no significant change in the skeletal muscle mass index (SMI) during NACRT. Of the 16 patients, nine (56%) were defined as sarcopenic before NACRT, and eight (50%) met the criteria for sarcopenic after NACRT. The SMI and total fat area were significantly associated with postoperative pancreatic fistula (POPF) (P=0.019 and P=0.007, respectively). The patients with sarcopenia had a shorter disease-free survival time and overall survival time in comparison to patients without sarcopenia (P=0.025 and P=0.115, respectively). In conclusion, NACRT for distal bile duct cancer did not significantly affect the body composition, or the immunological or nutritional indices. Sarcopenia after NACRT was significantly associated with early recurrence in patients with distal bile duct cancer who received NACRT.

The Site of Lymph Node Metastasis: A Significant Prognostic Factor in Pancreatic Ductal Adenocarcinoma.

While the presence and number of metastatic lymph nodes (LNs) are important prognostic factors for pancreatic ductal adenocarcinoma (PDAC), there is no recommendation to specify metastatic regional LN localization in the current staging system. The aim of this study was to evaluate the prognostic effect of regional metastatic LN localizations in PDAC. Metastatic sites of 101 consecutive PDAC patients who underwent pancreaticoduodenectomy were classified as peripancreatic, perigastric, hepatica communis, hepatoduodenal, and superior mesenteric artery. The frequency of metastasis in each region and the association between the presence of metastasis in each site and overall and disease-free survival were statistically analyzed. Eighty cases (79.2%) had peripancreatic, 7 (6.9%) had perigastric, 6 (5.9%) had hepatica communis, 7 (6.9%) had hepatoduodenal, and 4 (4%) had superior mesenteric artery LN metastasis. The overall and disease-free survival values were significantly shorter in patients with hepatoduodenal LN metastasis (log rank; p= 0.001, p=0.017, respectively). The presence of metastatic superior mesenteric artery LN was significantly associated with shorter disease-free survival in univariate analysis (p=0.017). Hepatoduodenal LN metastasis was an independent predictor of mortality (p=0.005) in multivariate analysis. The presence of hepatoduodenal LN metastasis is an independent poor prognostic factor for mortality. The presence of metastatic LN in the superior mesenteric artery region was significantly associated with shorter disease-free survival time, although not an independent predictor. We conclude that the metastatic regional LN sites, especially the hepatoduodenal region, have an impact on the prognosis, and should be included in synoptic pathology reports.

Cystatin-B Negatively Regulates the Malignant Characteristics of Oral Squamous Cell Carcinoma Possibly Via the Epithelium Proliferation/Differentiation Program.

Disturbance in the proteolytic process is one of the malignant signs of tumors. Proteolysis is highly orchestrated by cysteine cathepsin and its inhibitors. Cystatin-B (CSTB) is a general cysteine cathepsin inhibitor that prevents cysteine cathepsin from leaking from lysosomes and causing inappropriate proteolysis. Our study found that CSTB was downregulated in both oral squamous cell carcinoma (OSCC) tissues and cells compared with normal controls. Immunohistochemical analysis showed that CSTB was mainly distributed in the epithelial structure of OSCC tissues, and its expression intensity was related to the grade classification. A correlation analysis between CSTB and clinical prognosis was performed using gene expression data and clinical information acquired from The Cancer Genome Atlas (TCGA) database. Patients with lower expression levels of CSTB had shorter disease-free survival times and poorer clinicopathological features (e.g., lymph node metastases, perineural invasion, low degree of differentiation, and advanced tumor stage). OSCC cell models overexpressing CSTB were constructed to assess the effects of CSTB on malignant biological behaviors and upregulation of CSTB inhibited cell proliferation, migration, and invasion in vitro. Weighted gene correlation network analysis (WGCNA) and gene set enrichment analysis (GSEA) were performed based on the TCGA data to explore potential mechanisms, and CSTB appeared to correlate with squamous epithelial proliferation-differentiation processes, such as epidermal cell differentiation and keratinization. Moreover, in WGCNA, the gene module most associated with CSTB expression (i.e., the brown module) was also the one most associated with grade classification. Upregulation of CSTB promoted the expression levels of markers (LOR, IVL, KRT5/14, and KRT1/10), reflecting a tendency for differentiation and keratinization in vitro. Gene expression profile data of the overexpressed CSTB cell line were obtained by RNA sequencing (RNA-seq) technology. By comparing the GSEA enrichment results of RNA-seq data (from the OSCC models overexpressing CSTB) and existing public database data, three gene sets (i.e., apical junction, G2/M checkpoint, etc.) and six pathways (e.g., NOTCH signaling pathway, glycosaminoglycan degradation, mismatch repair, etc.) were enriched in the data from both sources. Overall, our study shows that CSTB is downregulated in OSCC and might regulate the malignant characteristics of OSCC via the epithelial proliferation/differentiation program.

MicroRNA-939 Promotes the Vitality of Human Breast Cancer Cells via Inhibition of E2F1/P73 Signaling

We assessed miR-939’s role in breast cancer (BC) and its molecular mechanism. PCR was performed to detect miRNA levels. Correlations between miR-939 and patients’ pathological information were analyzed. After transfection of E2F1 plasmid, P73 plasmid, si-E2F1, si-P73, miR-939 mimic or si-miR-939, cell proliferation and apoptosis were measured. The miR-939 target gene was proved by a luciferase assay. Protein and mRNA levels of E2F1 and P73 were detected by immunoblotting and PCR, and corresponding proliferation or apoptosis were assessed. MiR-939 expression was significantly increased in BC and associated with TNM staging, Ki-67 enhancement, and shorter disease-free survival time. In BC clinical samples, E2F1 expression is negatively correlated with miR-939 expressions. Overexpressing miR-939 stimulated growth but suppressed cell apoptosis. Functional analysis indicated E2F1 is the target gene of miR-939, and overexpression of miR-939 significantly downregulated E2F1 and P73. Silencing of E2F1 or P73 significantly promoted MDA-MB-231 cell proliferation and inhibited apoptosis. Overexpression of E2F1 plasmid or P73 plasmid significantly inhibited MDA-MB-231 cell proliferation but induced apoptosis. Transfection of P73 or E2F1 plasmid abolished miR-939’s effects on proliferation and apoptosis. miR-939 promotes breast cancer progression by downregulation of E2F1 to inhibit P73 pathway, thereby promoting proliferation and inhibiting apoptosis.

GIPC2 is an endocrine-specific tumor suppressor gene for both sporadic and hereditary tumors of RET- and SDHB-, but not VHL-associated clusters of pheochromocytoma/paraganglioma

Pheochromocytoma/paraganglioma (PPGL) is an endocrine tumor of the chromaffin cells in the adrenal medulla or the paraganglia. Currently, about 70% of PPGLs can be explained by germline or somatic mutations in several broadly expressed susceptibility genes including RET, VHL, and SDHB, while for the remaining, mainly sporadic cases, the pathogenesis is still unclear. Even for known susceptible genes, how mutations in these mostly ubiquitous genes result in tissue-specific pathogenesis remains unanswered, and why RET-mutated tumors almost always occur in the adrenal while SDHB-mutated tumors mostly occur extra-adrenal remains a mystery. By analyzing 22 sporadic PPGLs using SNP 6.0 genotyping arrays combined with expression profiling of 4 normal and 4 tumor tissues, we identified GIPC2, a gene located at 1p31.1 with preferential expression in adrenal and inducible by adrenal glucocorticoid, as a novel putative tumor suppressor gene for PPGLs. Copy number deletion and GIPC2 promoter hypermethylation but not GIPC2 mutation, accompanied with reduced GIPC2 expression, were observed in 39 of 55 PPGLs in our cohort. Examination of a published expression database consisting of 188 PPGLs found little GIPC2 expression in Cluster 1A (SDHx-associated) and Cluster 2A (NF1/RET-associated) tumors, but less pronounced reduction of GIPC2 expression in Cluster 1B (VHL-associated) and Cluster 2B/2C tumors. GIPC2 induced p27, suppressed MAPK/ERK and HIF-1ɑ pathways as well as cancer cell proliferation. Overexpressing GIPC2 in PC12 cells inhibited tumor growth in nude mice. We found GIPC2 interacted with the nucleoprotein NONO and both proteins regulated p27 transcription through the same GGCC box on p27 promoter. Significantly, low expression of both GIPC2 and p27 was associated with shorter disease-free survival time of PPGLs patients in the TCGA database. We found that PPGL-causing mutations in RET and in SDHB could lead to primary rat adrenal chromaffin cell proliferation, ERK activation, and p27 downregulation, all requiring downregulating GIPC2. Notably, the RET-mutant effect required the presence of dexamethasone while the SDHB-mutant effect required its absence, providing a plausible explanation for the tumor location preference. In contrast, the PPGL-predisposing VHL mutations had no effect on proliferation and GIPC2 expression but caused p53 downregulation and reduced apoptosis in chromaffin cells compared with wild-type VHL. Thus, our study raises the importance of cortical hormone in PPGL development, and GIPC2 as a novel tumor suppressor provides a unified molecular mechanism for the tumorigenesis of both sporadic and hereditary tumors of Clusters 1A and 2A concerning SDHB and RET, but not tumors of Cluster 1B concerning VHL and other clusters.

A qualitative transcriptional signature of recurrence risk for stages II-III gastric cancer patients after surgical resection.

Metastasis is the leading cause of recurrence in gastric cancer. However, the imaging techniques and pathological examinations for tumor metastasis have a high false-positive rate or a high false-negative rate, and many proposed that metastasis-related molecular biomarkers can hardly be validated in independent datasets. We propose to use significantly stable gene pairs with reversal relative expression orderings (REOs) between non-metastasis and metastasis gastric cancer samples as the metastasis-related gene pairs. Based on the REOs of these gene pairs, we developed a qualitative transcriptional signature for predicting the recurrence risk of stages II-III gastric cancer patients after surgical resection. A REOs-based signature, consisting of 19 gene pairs (19-GPS), was selected from 77 stages II-III gastric cancer patients and validated in two independent datasets. Samples in the high-risk group had shorter disease-free survival time and overall survival time than those in the low-risk group. Differentially expressed genes (DEGs) between the high- and low-risk groups classified by 19-GPS were highly reproducible comparing with those between lymph node metastasis and lymph node non-metastasis groups. Functional enrichment analysis showed that these DEGs were significantly enriched in metastasis-related pathways, such as PI3K-Akt and Rap1 signaling pathways. The multi-omics analyses suggested that the epigenetic and genomic features might cause transcriptional differences between two subgroups, which help to characterize the mechanism of gastric cancer metastasis. The signature could robustly identify patients at high recurrence risk after resection surgery, and the multi-omics analyses might aid in revealing the metastasis-related characteristics of gastric cancer.

MicroRNAs as Biomarkers in Canine Osteosarcoma: A New Future?

Sarcomas are frequent in dogs and canine species are excellent animal models for studying the human counterpart. However, osteosarcomas are a rare form of sarcoma with high death rates in humans and dogs. miRNAs are small endogenous RNAs that regulate gene expression post-transcriptionally. The discovery of miRNAs could give a contribute in the diagnosis and prognosis of different types of tumors in animal species, as already in humans. The differentiated expression of miRNAs is a frequent finding in cancers and is related to their pathogenesis in many cases. Most canine and human sarcomas show similar miRNA aberrations. Lower levels of miR-1 and miR-133b in canine osteosarcoma tissues were found to increase tumorigenesis through a higher expression of their target genes MET and MCL1. The overexpression of miR-9 promotes a metastatic phenotype in canine osteosarcomas and its capacity as a prognostic biomarker for the disease is currently being evaluated. MicroRNAs at the 14q32 locus could be used as prognostic biomarkers, since their decreased expression has been associated with poor prognosis in canine and human osteosarcomas. Furthermore, a decreased expression of miR-34a in osteosarcoma tumour cells has been associated with shorter disease-free survival times and its reintroduction as a synthetic prodrug shows good potential as a novel therapeutic target to fight the disease. Circulating miR-214 and miR-126 are significantly increased in a broad-spectrum cancer and have the ability to successfully predict the prognosis of dogs. However, further studies are needed to make the use of miRNAs as biomarkers a common practice.

PRMT5, regulated by lncRNA ZFAS1/miR-150-5p, promoted androgen-independent prostate cancer migration and invasion

IntroductionProstate cancer (PCa) is the most common male genitourinary malignancy in the world. The protein arginine methyltransferase 5 (PRMT5) is one of the main members of the type II PRMT family, which was reported to regulate androgen-dependent PCa cell proliferation. However, the upstream regulators of PRMT5 and its effects on androgen-independent PCa metastasis remained unclear. In the present study, we investigated whether PRMT5 could be a novel diagnostic marker and be used as a therapeutic target in PCa, to explore the possible molecular mechanism, and to understand the clinical importance of PRMT5 in PCa.Material and methodsThe present study evaluated PRMT5 expression levels in PCa and normal prostate samples using public datasets, including TCGA, GEPIA and GSE21032. Furthermore, CCK-8 assay, flow cytometer assay, and transwell assay were conducted to detect the roles of PRMT5. Luciferase reporter assay was used to determine the relationship among ZFAS1/miR-150-5p/PRMT5.ResultsOur results showed that PRMT5 was overexpressed in PCa samples. PRMT5 significantly promoted androgen-dependent PCa proliferation and cell cycle progression and suppressed cell apoptosis. However, PRMT5 did not affect androgen-independent PCa proliferation but it could significantly induce androgen-independent PCa metastasis. Knockdown of PRMT5 suppressed, whereas overexpression of PRMT5 induced, cell migration and invasion in androgen-independent DU145 and PC-3 cells. Moreover, our results showed that the ZFAS1/miR-150-5p axis regulated PRMT5 expression in PCa cells. Furthermore, the study showed that ZFAS1 and PRMT5 were overexpressed and miR-150-5p was down-regulated in PCa samples. Higher expression levels of ZFAS1 and PRMT5 were correlated with shorter disease-free survival time in PCa patients.ConclusionsThese results showed that PRMT5 may be a therapeutic target for PCa.

Overexpression of Ubiquitin-Specific Protease15 (USP15) Promotes Tumor Growth and Inhibits Apoptosis and Correlated With Poor Disease-Free Survival in Hepatocellular Carcinoma.

USP15 is a member of ubiquitin-specific proteases (USPs, the largest subfamily of deubiquitinases) and functions as a stabilize factor of target proteins in reversible ubiquitiantion progression. Dysregulated expression of USP15 has been observed in various cancers. However the expression profile and regulatory mechanism of USP15 in hepatocellular carcinoma (HCC) remains largely elusive. To exam the USP15 expression changes in the progression of HCC, we performed IHC analysis to test USP15 expression in a series of cancer-prone diseases including 2 normal liver tissues, 6 liver cirrhosis, 16 primary liver lesions and 15 metastases of hepatocellular carcinoma. The expression of USP15 was upregulated in various liver diseases in compared with normal tissue significantly (p < 0.05). Although no significant different of USP15 expression were discovered between cirrhotic tissue and primary tissue, its expression in HCC metastatic tissue was upregulated. Subsequently, we test the USP15 expression profile in a cohort of 66 HCC patients. USP15 expression was positively correlated with the recurrence of HCC significantly (p = 0.004). HCC patients with high USP15 expression had shorter disease free survival time in compare with those with low USP15 expression (56.9% VS 26.7%, P = 0.012). Subsequently, Cox multivariate analyses of clinical factors associated with disease free survival were performed and USP15 expression (p = 0.008) together with tumor size (p = 0.034) were proved to be independent predict factors in HCC. Then, we silenced USP15 expression in HCC cells and the results showed that downregulated USP15 expression resulting proliferation inhibition and apoptosis induction. In conclusion, our results suppose USP15 to be a potential target in HCC.

Doublecortin like Kinase-1 is Overexpressed in Breast Cancer Tissues and Correlated with Epithelial-mesenchymal Transition Markers

Background: Much of the current literature support, the idea that epithelial to mesenchymal transition (EMT) is the key mechanism by which tumor cells gain invasive and metastatic ability, as EMT enables separation of individual cells from the primary tumor mass as well as promote migration. After undergoing EMT, thereby enabling access to hematogenous or lymphatic routes of dissemination, tumor cells can extravasate into secondary organs and establish micro-metastases. Objective: The main target of this work was to study the relation between expression of Doublecortin-like kinase-1 (DCLK-1) and epithelial to mesenchymal transition markers (E-cadherin, vimentin and transforming growth factor-beta (TGF-β)) in breast cancer patients and assess its role in cancer prognosis. Materials and Methods: This study included 60 breast cancer patients and 40 healthy females as control group. Tumor tissues and adjacent normal breast tissues were collected from patients with breast cancer. A single venous blood sample was collected concurrently from breast cancer patients (Before surgery) and from the control group. Tissue expression of DCLK-1, E-cadherin and vimentin were evaluated in breast cancer tissues and normal breast tissues by real-time reverse transcription polymerase chain reaction (RT-PCR). Serum level of TGF-β was assayed by enzyme linked-immunossorbant assay. Results: According to the results of the present study DCLK-1 is highly expressed in breast cancer tissues compared to normal breast tissues. Overexpression of DCLK-1 was significantly correlated with higher tissues Vimentin expression, increased serum TGF-β and lower tissues E-cadherin expression. Kaplan-Meier survival curves for breast cancer patients revealed that, patients with elevated tissue DCLK-1 and Vimentin expression and higher serum TGF-β were significantly associated with poor prognosis in primary breast cancer patients. However, patients with lower tissue E-Cadherin expression had shorter disease free survival time than patients with higher levels. Conclusion: DCLK-1 was significantly increased in breast cancer tissues in comparison to normal breast tissues and its overexpression was significantly correlated with EMT markers and poor prognosis in breast cancer patients. Further prospective studies using greater numbers of patients are required to confirm our findings.

Expression patterns of programmed death-1 and programmed death-1 ligand-1 on T cells in gastric cancer.

The aim of the present study was to evaluate programmed death-1 (PD-1) and programmed death-1 ligand-1 (PD-L1) expression in gastric carcinoma and to assess their effect on survival rate. A total of 170 surgically resected specimens were obtained from patients diagnosed with gastric carcinoma at St. Vincents Hospital, The Catholic University of Korea. Paraffin tissue sections from tissue microarray blocks were subjected to immunohistochemical analysis of PD-1 and PD-L1. In addition, PD-1 expression on CD4+ and CD8+ T cells isolated from peripheral blood mononuclear cells and gastric cancer tissues was evaluated by multicolor flow cytometry. PD-1 and PD-L1 were expressed in 30.0 and 60.5% of the gastric cancer tissues, respectively. The expression of PD-L1 was higher in patients with advanced T (P=0.035) and Tumor, Node and Metastasis stage (P=0.05). The patients with positive PD-L1 expression had shorter disease-free survival time than those without PD-L1 expression (P=0.005). Additionally, PD-L1 expression was significantly associated with poor prognosis (P=0.015). PD-1 and PD-L1 expression levels were significantly higher on CD8+ T cells than on CD4+ T cells (P<0.001). The data of the present study suggested that PD-L1 expression may be an independent indicator of poor prognosis in patients with gastric cancer. Furthermore, PD-L1 expression may play a role in immune evasion of gastric cancer.

Novel K6-K14 keratin fusion enhances cancer stemness and aggressiveness in oral squamous cell carcinoma.

Keratin intermediate filament (IF) is one component of cellular architectures, which provides necessary mechanical support to conquer environmental stresses. Recent findings reveal its involvement in mechano-transduction and the associated stem cell reprogramming, suggesting the possible roles in cancer development. Here, we report t(12;17)(q13.13;q21.2) chromosomal rearrangement as the most common fusion event in OSCC, resulting in a variety of inter-keratin fusions. Junction site mapping verified 9 in-frame K6-K14 variants, three of which were correlated with lymph node invasion, late tumor stages (T3/T4) and shorter disease-free survival times. When expressed in OSCC cells, those fusion variants disturbed wild-type K14 organization through direct interaction or aggregate formation, leading to perinuclear structure loss and nuclear deformation. Protein array analyses showed the ability of K6-K14 variant 7 (K6-K14/V7) to upregulate TGF-β and G-CSF signaling, which contributed to cell stemness, drug tolerance, and cell aggressiveness. Notably, K6-K14/V7-expressing cells easily adapted to a soft 3-D culture condition in vitro and formed larger, less differentiated tumors in vivo. In addition to the anti-mechanical-stress activity, our data uncover oncogenic functionality of novel keratin filaments caused by gene fusions during OSCC development.

Silencing of HAS2-AS1 mediates PI3K/AKT signaling pathway to inhibit cell proliferation, migration, and invasion in glioma.

Hyaluronan synthase 2 (HAS2)-AS1 (natural antisense transcript of HAS2) functions as oncogenic long noncoding RNA (lncRNA) in oral squamous cell carcinoma, breast cancer, and osteosarcoma. The role of HAS2-AS1 in glioma remains unknown. In our research, HAS2-AS1 expression was elevated in glioma tissues compared with normal brain tissues. Moreover, high levels of HAS2-AS1 expression was observed in patients with glioma with high WHO grade (III-IV) or large tumor size ( > 4 cm). The survival analysis from The Cancer Genome Atlas showed glioma cases with high HAS2-AS1 expression that had shorter disease-free survival time and overall survival time than those with low HAS2-AS1 expression. In vitro studies suggested that knocking down HAS2-AS1 expression inhibited glioma cell viability, migration, and invasion through phosphoinositide 3-kinase/protein kinase B signaling pathway. In conclusion, HAS2-AS1 may be considered as a predictor for clinical outcome and a potential therapeutic target in glioma.

High p16 Expression Is Associated with Malignancy and Shorter Disease-Free Survival Time in Solitary Fibrous Tumor/Hemangiopericytoma.

Objective Solitary fibrous tumors (SFT) and hemangiopericytomas (HPC) are now classified along a single spectrum of fibroblastic mesenchymal tumors with NAB2-STAT6 fusion. This fusion acts as a driver mutation that constitutively activates EGR1, which is known to be involved in the p16 pathway. Overexpression of p16 is associated with malignancy and worse prognosis in multiple mesenchymal tumors. The authors sought to investigate p16 immunoexpression in association with malignancy and prognosis of SFT/HPC tumors. Design Twenty-three SFT/HPC tumors (central nervous system [CNS]: 12, non CNS: 11) diagnosed at our institution from 2002 to 2016 were assigned into 3 grades. Data from microarray immunohistochemistry for STAT6, synaptophysin, CD56, chromogranin, SST2A, EGR1, Ki67, and p16, grade and survival were analyzed. Results CNS SFT/HPCs tend to be malignant (grade 3; 67 vs. 18%, p = 0.036) and more likely to express synaptophysin (33 vs. 0%, p = 0.035) than non CNS tumors. Overexpression of p16 (immunopositivity ≥ 50% tumor cells) was associated with malignant (grade 3) tumors, and has a sensitivity of 70% (7/10), and a specificity of 77% (10/13), as a predictive marker for malignancy. SFT/HPC patients with low p16 expression demonstrated significantly longer disease-free survival time (median survival > 113 months) than those with high p16 expression (median survival = 30 months, p = 0.045). Conclusions SFT/HPCs in the CNS are more likely to be malignant than the tumors in other sites. High p16 expression is also associated with malignancy and shorter disease-free survival time in SFT/HPC tumors in our study cohort. Clinically, p16 overexpression can be used as predictive marker for malignancy and prognosis and a possible therapeutic target.

Prognostic value of zymographic gelatinase activity of MMP-2 and MMP-9 in tumor recurrence of canine intrascrotal hemangiomas

There is limited information about intrascrotal hemangioma tumors and their gelatinase activity in the dogs. This study was planned to identify the serum gelatinases in male dogs affected by intrascrotal hemangioma and determine their prognostic value in tumor recurrence. Ten dogs with intrascrotal hemangioma were diagnosed among a total of 65 testicular excision samples. According to the incidence of recurrence, the patients divided into two groups: tumors with recurrence and tumors with no-recurrence. Serum gelatinase activity was assayed by semi-quantitative zymography to determinate their prognostic value in canine intrascrotal hemangioma. Both latent and active MMP-9 and only latent MMP-2 appeared in the gels. Gelatinases showed a significant higher serum activity in dogs with hemangioma than those of the normal dogs. There was a significant association between increased serum activity of gelatinase A and gelatinase B and tumor recurrence. The dogs with hemangioma had a shorter disease-free survival time; however, multivariate analysis showed that serum activity of MMP-2 and MMP-9 was not an independent prognostic factor. According to the findings, MMP-2 and MMP-9 may cause a progressive angiogenesis in canine intrascrotal hemangioma and tumor recurrence subsequently. Our results showed that serum activity of MMP-2 and MMP-9 may be used as a non-invasive prognostic marker for tumor recurrence prediction in dogs affected by intrascrotal hemangioma.

Clinical significance of NS1-BP expression in esophageal squamous cell carcinoma

Objective: To investigate the clinical significance of NS1-BP expression in patients with esophageal squamous cell carcinoma (ESCC), and to study the roles of NS1-BP in proliferation and apoptosis of ESCC cells. Methods: A total of 98 tumor tissues and 30 adjacent normal tissues from 98 ESCC patients were used as study group and control group, and these samples were collected in Sun Yat-Sen University Cancer Center between 2002 and 2008. In addition, 46 ESCC tissues which were collected in Cancer Institute and Hospital of Tianjin Medical University were used as validation group. Expression of mucosal NS1-BP was detected by immunohistochemistry. Kaplan-Meier curve and log-rank test were used to analyze the survival rate. Multivariate Cox proportional hazard model was used to analyze the prognostic factors. Furthermore, NS1-BP was over expressed or knocked down in ESCC cells by transient transfection. Protein levels of c-Myc were detected by western blot. Cell viability and apoptosis was analyzed by MTT assay and flow cytometry. Results: Among all of tested samples, NS1-BP were down-regulated in 9 out of 30 non-tumorous normal esophageal tissues (30.0%) and 85 out of 144 ESCC tissues (59.0%), respectively, showing a statistically significant difference (P=0.012). In the study group, three-year disease-free survival rate of NS1-BP high expression group (53.2%) was significantly higher than that of NS1-BP low expression group (27.6%; P=0.009). In the validation group, the three-year disease-free survival rates were 57.8% and 25.5% in NS1-BP high and low levels groups, respectively, showing a similar results (P=0.016). Importantly, multivariate analyses showed that low expression of NS1-BP was an independent predictor for chemoradiotherapy sensitivity and shorter disease-free survival time in ESCC patients(P<0.05 for all). Furthermore, overexpressed NS1-BP in TE-1 cells repressed c-Myc expression, inhibited cell proliferation and promoted apoptosis. In contrast, knockdown NS1-BP in KYSE510 cells induced c-Myc expression, increased cell proliferation and repressed apoptosis. Conclusions: NS1-BP is an independent favorable prognostic factor in ESCC. It inhibits cell proliferation and enhances cell apoptosis via repressing c-Myc. Targeting NS1-BP may be a new therapeutic strategy for ESCC patients.

Abstract A21: Association between polymorphisms in inflammatory response related-genes and the susceptibility, progression, and prognosis of gastric cancer

Abstract Introduction: The chronic inflammatory microenvironment in the stomach has been described as a critical component for both tumor initiation and progression. Furthermore, genetic variants have been shown to influence the inter-individual variations in the inflammatory response. Therefore, we aimed to investigate whether polymorphisms in inflammatory response related-genes were associated with risk for gastric tumor development, clinical outcomes, and overall and disease-free survival of this disease in a Brazilian population sample. Methods: Sixteen selected genetic variants in eleven genes (COX-2, OGG1, TNFB, TNFA, HSPA1L, HSPA1B, VEGFA, IL17F, LGALS3, PHB, and TP53) were genotyped in 262 control individuals and 178 gastric cancer patients. Genetic association analyses were investigated in different models (Genotype, Allele, Dominant, and Recessive) in both total sample (N=178) and stratified for the diffuse histologic subtype based on Lauren´s classification (N=112). We also calculated the linkage disequilibrium among the polymorphisms and the haplotype associations were carried out using Haploview and PLINK softwares. Results: Regarding the susceptibility genetic markers, we found that rs1042522 (TP53) Pro allele carriers presented about 2-fold higher risk for developing gastric cancer in a multivariate analysis and this association was even stronger when analyzing only cases with the diffuse subtype. We also found that CTC haplotype (composed by rs699947, rs833061, and rs2010963 of VEGFA) was associated with gastric malignancy. On the other hand, the presence of A allele of rs699947 (VEGFA) was associated with a protection against developing this disease. Regarding the disease progression, the following polymorphisms/haplotypes were able to predict outcomes associated with a worse aggressiveness: rs689466 (COX-2); rs1052133 (OGG1); rs699947, rs833061, and rs2010963 (VEGFA); rs4644 (LGALS3); rs1042522 (TP53); GG haplotype (TNFB/TNFA); ACG and CTC haplotype (VEGFA) and GT haplotype (rs689466 and rs5275 of COX-2 gene). On the other hand, other variants were associated with better outcomes: rs5275 (COX-2); rs2227956 (HSPA1L), and rs3025039 (VEGFA). We also observed that the rs909253 (TNFB) polymorphism was able to predict a better outcome only when stratifying for the individuals diagnosed with the diffuse subtype. Finally, regarding the impact on prognosis, rs909253 (TNFB) was associated with a favorable prognosis when analyzing both the overall and disease-free survivals while rs4644 (LGALS3) His allele carriers presented a worse prognosis with shorter disease-free survival time. Conclusions: These results helped us to clarify the potential role of these polymorphisms in genes involved in the modulation of the inflammatory response in the pathogenesis of gastric malignancy, highlighting that the host genetic variants might act together with other factors to influence the susceptibility, progression, and prognosis of gastric cancer. Citation Format: Tatiane K. Furuya, Carlos E. Jacob, Michele T. Tomitão, Lizeth C. Cordoba-Camacho, Marcus K. Ramos, José Eluf-Neto, Venâncio A. Alves, Bruno Zilberstein, Ivan Cecconello, Ulysses Ribeiro-Junior, Roger Chammas. Association between polymorphisms in inflammatory response related-genes and the susceptibility, progression, and prognosis of gastric cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A21.

KIF11 is required for proliferation and self-renewal of docetaxel resistant triple negative breast cancer cells.

Development of chemoresistance remains a major hurdle for triple negative breast cancer treatment. Previous studies suggest that CD44+/CD24- cells, subpopulation of cancer stem cells with self-renewing and tumor-initiating capacities, are partly responsible for chemoresistance and therapeutic failure of triple negative breast cancer. Therefore, novel agents that target cancer stem cells (CSCs) may improve the clinical outcome. KIF11 (kinesin family member 11), overexpressed in many cancer cells, is a molecular motor protein that plays essential role in mitosis. In this study, we assess its role in docetaxel resistant triple negative breast cancer (TNBC). We found that the expression of KIF11 was significantly increased in CD44+/CD24- subpopulation of docetaxel resistant TNBC cells. Knockdown of KIF11 resulted in a significant decrease in the percentage of CSCs and mammosphere formation. KIF11 knockdown also inhibits cell growth and induces cell cycle G2/M arrest followed by cell mitosis and apoptosis. Further docetaxel resistant TNBC xenograft models demonstrated that KIF11 inhibitor exerts growth inhibitory effect in vivo. Of note, we also found that KIF11 was highly expressed in TNBC and its expression was correlated with shorter disease free survival time. All these data indicate that KIF11 is critical for proliferation and self-renewal in TNBC tumor cells in vitro and in vivo, suggesting that KIF11 may be a promising therapeutic target for treating chemoresistant TNBC.

An autocrine inflammatory forward-feedback loop after chemotherapy withdrawal facilitates the repopulation of drug-resistant breast cancer cells

Stromal cells, infiltrating immune cells, paracrine factors and extracellular matrix have been extensively studied in cancers. However, autocrine factors produced by tumor cells and communications between autocrine factors and intracellular signaling pathways in the development of drug resistance, cancer stem-like cells (CSCs) and tumorigenesis have not been well investigated, and the precise mechanism and tangible approaches remain elusive. Here we reveal a new mechanism by which cytokines produced by breast cancer cells after chemotherapy withdrawal activate both Wnt/β-catenin and NF-κB pathways, which in turn further promote breast cancer cells to produce and secrete cytokines, forming an autocrine inflammatory forward-feedback loop to facilitate the enrichment of drug-resistant breast cancer cells and/or CSCs. Such an unexpected autocrine forward-feedback loop and CSC enrichment can be effectively blocked by inhibition of Wnt/β-catenin and NF-κB signaling. It can also be diminished by IL8-neutralizing antibody or blockade of IL8 receptors CXCR1/2 with reparixin. Administration of reparixin after chemotherapy withdrawal effectively attenuates tumor masses in a human xenograft model and abolishes paclitaxel-enriched CSCs in the secondary transplantation. These results are partially supported by the latest clinical data set. Breast cancer patients treated with chemotherapeutic drugs exhibited poor survival rate (66.7 vs 282.8 months, P=0.00071) and shorter disease-free survival time if their tumor samples expressed high level of IL8, CXCR1, CXCR2 genes and Wnt target genes. Taken together, this study provides new insights into the communication between autocrine niches and signaling pathways in the development of chemotherapy resistance and CSCs; it also offers a tangible approach in breast cancer treatment.