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Supplementary Table 1 from Clinical Significance of miR-146a in Gastric Cancer Cases

Purpose: The profiles of microRNAs change significantly in gastric cancer. MiR-146a is reported to be a tumor suppressor in pancreatic cancer, breast cancer, and prostate cancer. We investigated the clinical significance of miR-146a in gastric cancer, in particular focusing on hypothetical miR-146a target genes, such as epidermal growth factor receptor (EGFR) and interleukin-1 receptor-associated kinase (IRAK1).Experimental Design: We examined miR-146a levels in 90 gastric cancer samples by q-real-time (qRT)–PCR and analyzed the association between miR-146a levels and clinicopathologic factors and prognosis. The regulation of EGFR and IRAK1 by miR-146a was examined with miR-146a–transfected gastric cancer cells. Moreover, we analyzed the association between miR-146a levels and the G/C single nucleotide polymorphism (SNP) within pre-miR-146a seed sequences in 76 gastric cancer samples, using direct sequencing of genomic DNA.Results: In 90 clinical samples of gastric cancer, miR-146a levels in cancer tissues were significantly lower than those in the corresponding noncancerous tissue (P < 0.001). Lower levels of miR-146a were associated with lymph node metastasis and venous invasion (P < 0.05). Moreover, a lower level of miR-146a was an independent prognostic factor for overall survival (P = 0.003). Ectopic expression of miR-146a inhibited migration and invasion and downregulated EGFR and IRAK1 expression in gastric cancer cells. In addition, G/C SNP within the pre-miR-146a seed sequence significantly reduced miR-146a levels in the GG genotype compared with the CC genotype.Conclusions:MiR-146a contains an SNP, which is associated with mature miR-146a expression. MiR-146a targeting of EGFR and IRAK1 is an independent prognostic factor in gastric cancer cases. Clin Cancer Res; 17(13); 4277–84. ©2011 AACR.

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Supplementary Tables S1-S2 PDF file - 41K, CRC Patient characteristics in training set (2000-2004) and validation set (2005-2008) (S1); Gene list of GSEA analysis (S2).

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Supplementary Figure Legends PDF file - 49K, Legend for Supplementary Figures S1-S9

Circulating tumor cells (CTC) in blood have attracted attention both as potential seeds for metastasis and as biomarkers. However, most CTC detection systems might miss epithelial–mesenchymal transition (EMT)-induced metastatic cells because detection is based on epithelial markers. First, to discover novel markers capable of detecting CTCs in which EMT has not been repressed, microarray analysis of 132 colorectal cancers (CRC) from Japanese patients was conducted, and 2,969 genes were detected that were overexpressed relative to normal colon mucosa. From the detected genes, we selected those that were overexpressed CRC with distant metastasis. Then, we analyzed the CRC metastasis-specific genes (n = 22) to determine whether they were expressed in normal circulation. As a result, PLS3 was discovered as a CTC marker that was expressed in metastatic CRC cells but not in normal circulation. Using fluorescent immunocytochemistry, we validated that PLS3 was expressed in EMT-induced CTC in peripheral blood from patients with CRC with distant metastasis. PLS3-expressing cells were detected in the peripheral blood of approximately one-third of an independent set of 711 Japanese patients with CRC. Multivariate analysis showed that PLS3-positive CTC was independently associated with prognosis in the training set (n = 381) and the validation set [n = 330; HR = 2.17; 95% confidence interval (CI) = 1.38–3.40 and HR = 3.92; 95% CI = 2.27–6.85]. The association between PLS3-positive CTC and prognosis was particularly strong in patients with Dukes B (HR = 4.07; 95% CI = 1.50–11.57) and Dukes C (HR = 2.57; 95% CI = 1.42–4.63). PLS3 is a novel marker for metastatic CRC cells, and it possesses significant prognostic value. Cancer Res; 73(7); 2059–69. ©2012 AACR.

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Supplementary Figures S1-S9 PDF file - 984K, Representative photomicrographs of tissue sections immunostained for PLS3 (S1); The clinical significance of PLS3 expression in a second independent set of 110 primary colorectal cancer samples (S2); PLS3 expression profiles in various tissues, including peripheral blood-derived cells (S3); EMT induction in CaR-1 cells by TGF-beta1 (S4); PLS3 mRNA expression in LoVo cells expressing stem cell makers (S5); Immunocytochemical staining of PLS3 in rectal cancer cell line CaR-1 and peripheral blood mononuclear cells (S6); Validation of PLS3 expression in PB after recurrence (S7); Survival curves of Dukes stage B CRC patients based on the level of CK19/CK20 mRNA expression in PB (S8); PLS3 expression profiles in several cancer cell lines (S9)

The functional impact of recently discovered long noncoding RNAs (ncRNAs) in human cancer remains to be clarified. One long ncRNA which has attracted attention is the Hox transcript antisense intergenic RNA termed HOTAIR, a long ncRNA expressed from the developmental HOXC locus located on chromosome 12q13.13. In cooperation with Polycomb complex PRC2, the HOTAIR long ncRNA is reported to reprogram chromatin organization and promote breast cancer metastasis. In this study, we examined the status and function of HOTAIR in patients with stage IV colorectal cancer (CRC) who have liver metastases and a poor prognosis. HOTAIR expression levels were higher in cancerous tissues than in corresponding noncancerous tissues and high HOTAIR expression correlated tightly with the presence of liver metastasis. Moreover, patients with high HOTAIR expression had a relatively poorer prognosis. In a subset of 32 CRC specimens, gene set enrichment analysis using cDNA array data revealed a close correlation between expression of HOTAIR and members of the PRC2 complex (SUZ12, EZH2, and H3K27me3). Our findings suggest that HOTAIR expression is associated with a genome-wide reprogramming of PRC2 function not only in breast cancer but also in CRC, where upregulation of this long ncRNA may be a critical element in metastatic progression. Cancer Res; 71(20); 6320–6. ©2011 AACR.

Background: There is no guideline for hearing compensation after temporal bone resection. This study aimed to retrospectively analyze surgical cases with reconstruction for hearing preservation after temporal bone malignancy resection and propose a new alternative to compensate for hearing loss. Methods: We retrospectively reviewed the medical records of 30 patients who underwent lateral temporal bone surgery for temporal bone malignancy at our institution and examined their hearing abilities after surgery. Result: The hearing outcomes of patients with an external auditory meatus reconstruction varied widely. The mean postoperative air–bone gap at 0.5, 1, 2, and 4 kHz ranged from 22.5 dB to 71.25 dB. On the other hand, the average difference between the aided sound field thresholds with cartilage conduction hearing aid and bone conduction thresholds at 0.5, 1, 2, and 4 kHz ranged from −3.75 to 41.25. More closely located auricular cartilage and temporal bone resulted in smaller differences between the aided sound field and bone conduction thresholds. Conclusions: There is still room for improvement of surgical techniques for reconstruction of the auditory meatus to preserve hearing after temporal bone resection. The cartilage conduction hearing aid may provide non-invasive postoperative hearing compensation after lateral temporal bone resection.

Objective En bloc and margin-negative surgical resection seems to offer the best prognosis for patients with temporal bone squamous cell carcinoma (TB-SCC). In this study, we summarize the outcomes of surgical cases of advanced TB-SCC (T3–T4) that were managed in two institutions, with an accompanying description of the surgical procedure that was utilized: modified subtotal temporal bone resection (STBR), which involves the en bloc removal of the temporal bone including or transecting the otic capsule. Design This is a case series study with chart review. Setting The study was conducted at two academic tertiary care medical centers. Participants Chart information was collected for all patients who underwent surgical resection of advanced TB-SCC between July 1998 and February 2019. The resulting dataset contained 43 patients with advanced TB-SCC who underwent en bloc resection during the review period. Tumor staging followed the modified Pittsburgh classification. Disease-specific survival (DSS) rates were calculated according to the Kaplan–Meier method. Main Outcome Measure This study shows disease-specific 5-year DSS rate. Results The 5-year DSS rate of the cases who underwent en bloc resection was 79.7%. En bloc lateral temporal bone resection was employed in a total of 25 cases (DSS: 79.0%). En bloc modified STBR was utilized in 18 cases (DSS: 81.7%). Conclusion En bloc margin-negative resection is a reliable treatment strategy for advanced TB-SCC. Modified STBR can be a treatment option for TB-SCC without marked posterior extension.

External auditory canal squamous cell carcinoma (EACSCC) is an extremely rare and aggressive malignancy. Due to its rarity, the molecular and genetic characteristics of EACSCC have not yet been elucidated. To reveal the genetic alterations of EACSCC, we performed whole exome sequencing (WES) on 11 primary tumors, 1 relapsed tumor and 10 noncancerous tissues from 10 patients with EACSCC, including 1 with a rare case of synchronous bilateral EACSCC of both ears. WES of the primary tumor samples showed that the most frequently mutated gene is TP53 (63.6%). In addition, recurrent mutations in CDKN2A, NOTCH1, NOTCH2, FAT1 and FAT3 were detected in multiple samples. The mutational signature analysis of primary tumors indicated that the mutational processes associated with the activation of apolipoprotein B mRNA‐editing enzyme catalytic polypeptide‐like (APOBEC) deaminases are the most common in EACSCC, suggesting its similarity to SCC from other primary sites. Analysis of arm‐level copy number alterations detected notable amplification of chromosomes 3q, 5p and 8q as well as deletion of 3p across multiple samples. Focal chromosomal aberrations included amplifications of 5p15.33 (ZDHHC11B) and 7p14.1 (TARP) as well as deletion of 9p21.3 (CDKN2A/B). The protein expression levels of ZDHHC11B and TARP in EACSCC tissues were validated by immunohistochemistry. Moreover, WES of the primary and relapsed tumors from a case of synchronous bilateral EACSCC showed the intrapatient genetic heterogeneity of EACSCC. In summary, this study provides the first evidence for genetic alterations of EACSCC. Our findings suggest that EACSCC mostly resembles other SCC., As the genetic characteristics of external auditory canal squamous cell carcinoma (EACSCC) are not yet elucidated due to its rarity, we performed whole exome sequencing and copy number analysis in EACSCC samples. The genetic alterations of EACSCC mostly resemble other SCC, although the novel amplified loci that harbor oncogenes were found.

Objective: Sacrificing the auricle has severe postoperative impacts on patients with temporal bone malignancy who have undergone en bloc temporal bone resection. The auricular complications following en bloc temporal bone resection remain unclear. Therefore, this study aimed to examine auricular complications following en bloc temporal bone resection and reveal the associated risk factors. Study Design: Case series with chart review. Setting: Single tertiary care medical center Participants: Our study includes all cases who underwent en bloc temporal bone resection with preserved auricle for temporal bone malignancy from January 1993 to July 2020. The resulting dataset contained 64 patients during the review period. Main outcome measures: We examined the relationship of auricular complications with surgical procedures and patient profiles. Results: Three of 64 cases (4.7%) showed postoperative auricular complication. The cause in all three cases was venous congestion. Postoperative bloodletting to relieve the congestion effectively salvaged the auricle in two of these cases. Insufficient bloodletting to relieve the auricular congestion in the remaining case led to auricular necrosis. Pre-/infra-auricular lymph node dissection is correlated with auricular complications with statistical significance. Conclusions: Temporal bone resection causes auricular congestion due to vascular insufficiency, particularly insufficient venous outflow. An immediate and continuous application of bloodletting can improve venous drainage after temporal bone resection to prevent auricle necrosis.

Objective: The extreme rarity of temporal bone squamous cell carcinoma (TB-SCC) has delayed the accumulation of high-quality clinical evidence. Our objective here was to explore anatomical factors associated with the prognosis of T4 TB-SCC cases. Study Design: Case series with chart review. Setting: Two academic tertiary care medical centers. Subjects and Methods: The medical records of all TB-SCC cases were retrospectively reviewed in two institutions. The resulting data set contained 30 cases of primary T4 cancer eligible for initial definitive (curative) treatment. Disease-specific survival was calculated according to the Kaplan–Meier method. Cox proportional hazards model was used to identify anatomical prognosis factors. Results: The disease-specific 5-years survival rate of 30 cases of T4 TB-SCC was 53.9%. The tumor invasion to the pterygoid muscle, posterior fossa dura, and sigmoid sinus and destruction of the ossicles were associated with poor prognosis in univariate analysis. The multivariate analysis reveals that the invasion of the ossicles, posterior fossa dura, and sigmoid sinus is an independent prognostic factor [hazard ratio (HR): 4.528 (95% CI: 1.161–17.658), p = 0.030; HR: 5.135 (95% CI: 1.616–16.315), p = 0.006; HR: 4.292 (95% CI: 1.385–13.303), p = 0.012]. The invasion of the carotid canal, petrous apex, middle fossa dura, otic capsule, pterygoid muscle, and middle ear had a high HR (HR > 2). The more invaded anatomical factors present in patients resulted in a poorer patient disease-specific prognosis, with a statistically significant difference. Conclusions: Assessing which anatomical structures are susceptible to invasion by tumors may be important for predicting TB-SCC patient prognosis and selecting appropriate treatment planning, especially surgical intervention. In addition to previously reported factors, the destruction of the ossicles in the middle ear cavity can be an anatomical prognosis factor.

Objective The usefulness of pretreatment measurement of SCC antigen in patients with head and neck SCC is still controversial. Our aim of this study was to evaluate the clinical usefulness of serum SCC antigen, SCCA1 and SCCA2 in the management of patients with head and neck SCC. Methods Serum samples for the analysis of SCCA1, SCCA2 and SCC antigen were taken from head and neck SCC patients before treatment. Serum SCC antigen was assayed with a solid phase immunoradiometric assay. The SCCA1 and SCCA2 protein level was determined by a sandwich ELISA. Results Fifty-two of 96 cases (54%) showed evaluated serum SCC antigen levels above the upper limit. The serum SCCA2 level was significantly higher in the head and neck SCC patients than in control group, whereas there were no significant differences in the serum SCCA1 level between head and neck SCC patients and control group. 72% of head and neck SCC patients demonstrated SCCA2 levels higher than 0.15, whereas 68% of the control subjects had SCCA2 levels less than 0.15. Conclusion The serum SCCA2 levels were increased during the progression of cancer and might be a useful tool for the management of head and neck SCC.

Loss of Heterozygosity (LOH) is commonly considered to be one of a reason when some genes lose their function. Numbers of tumor suppressor genes are existing on the LOH lesion and chromosome 16q24 had been reported as a LOH region in gastric cancer. Little is known about what kind of tumor suppressor genes locates around the position. F-box protein, (FBXO31) is a candidate tumor suppressor gene encoded in chromosome 16q24.3 and LOH of the gene was reported in breast cancer, hepatocellular carcinoma and ovarian cancer but the status of FBXO31 was not analyzed in gastric cancer so far. One hundred twenty-seven pairs of tumor and corresponding normal tissue specimens collected from gastric cancer patients who underwent gastrectomy. Total RNAs were extracted from those samples and the expression of FBXO31 was investigated using real time quantitative RT-PCR analysis. Patients were classified into FBXO31 high expression group and low expression group. Clinicopahological factors were compared between the two groups and importance of FBXO31 was investigated. The standardized expression of FBXO31 was not significantly different between tumor (0.43 ± 0.46) and the corresponding 0.49 ± 0.55 in normal tissue (p = 0.39). Two years survival rate was 77% in FBXO31 high expression group and 54% in low expression group however the chance of survival rate of high expression group was dropped in 5 years (Wilcoxon p = 0.01). Clinicopathological factors were compared between the two groups and peritoneal dissemination was observed significantly higher in FBXO31 low expression group than did in high expression group (p = 0.0398). In order to predict existence of peritoneal dissemination of gastric cancer before surgery, FBXO31 may become a favorite marker for the low risk of peritoneal dissemination.

Reports of drug-induced interstitial pneumonia caused by Cetuximab have been increasing. Pneumocystis pneumonia is important as a differential diagnosis of drug-induced interstitial pneumonia. We report herein on a 64-year-old man with pneumocystis pneumonia after cetuximab-based bioradiotherapy for laryngeal cancer. After radiotherapy, the patient developed multi-drug resistant pneumonia. Chest CT imaging revealed diffuse ground-glass opacities in the lung field. He was diagnosed as having pneumocystis pneumonia based on the bronchoalveolar lavage (BAL) findings, and then his symptoms improved after treatment with Trimethoprim/Sulfamethoxazole. It is important to assess the risk factor for pneumocystis pneumonia for early its detection and treatment.

The identification of prognostic biomarkers and their underlying mechanisms of action remain of great interest in breast cancer biology. Using global miRNA profiling of 71 lymph node-negative invasive ductal breast cancers and 5 normal mammary epithelial tissues, we identified miR-449a to be highly overexpressed in the malignant breast tissue. Its expression was significantly associated with increased incidence of patient relapse, decreased overall survival, and decreased disease-free survival. In vitro, miR-449a promoted breast cancer cell proliferation, clonogenic survival, migration, and invasion. By utilizing a tri-modal in silico approach for target identification, Cysteine-Rich Protein 2 (CRIP2; a transcription factor) was identified as a direct target of miR-449a, corroborated using qRT-PCR, Western blot, and luciferase reporter assays. MDA-MB-231 cells stably transfected with CRIP2 demonstrated a significant reduction in cell viability, migration, and invasion, as well as decreased tumor growth and angiogenesis in mouse xenograft models. Our data revealed that overexpression of miR-449a suppresses CRIP2, which then affects the tumor vasculature, likely via NF-κB/p65 complex-mediated transcription of VEGF. These finding define an oncogenic function of miR-449a in human breast cancer, and highlight the importance of this pathway in driving aggressive behaviour.

PICT1 (also known as GLTSCR2) is considered a tumor suppressor because it stabilizes phosphatase and tensin homolog (PTEN), but individuals with oligodendrogliomas lacking chromosome 19q13, where PICT1 is located, have better prognoses than other oligodendroglioma patients. To clarify the function of PICT1, we generated Pict1-deficient mice and embryonic stem (ES) cells. Pict1 is a nucleolar protein essential for embryogenesis and ES cell survival. Even without DNA damage, Pict1 loss led to p53-dependent arrest of cell cycle phase G(1) and apoptosis. Pict1-deficient cells accumulated p53, owing to impaired Mdm2 function. Pict1 binds Rpl11, and Rpl11 is released from nucleoli in the absence of Pict1. In Pict1-deficient cells, increased binding of Rpl11 to Mdm2 blocks Mdm2-mediated ubiquitination of p53. In human cancer, individuals whose tumors express less PICT1 have better prognoses. When PICT1 is depleted in tumor cells with intact P53 signaling, the cells grow more slowly and accumulate P53. Thus, PICT1 is a potent regulator of the MDM2-P53 pathway and promotes tumor progression by retaining RPL11 in the nucleolus.

Purpose: The profiles of microRNAs change significantly in gastric cancer. MiR-146a is reported to be a tumor suppressor in pancreatic cancer, breast cancer, and prostate cancer. We investigated the clinical significance of miR-146a in gastric cancer, in particular focusing on hypothetical miR-146a target genes, such as epidermal growth factor receptor (EGFR) and interleukin-1 receptor-associated kinase (IRAK1). Experimental Design: We examined miR-146a levels in 90 gastric cancer samples by q-real-time (qRT)–PCR and analyzed the association between miR-146a levels and clinicopathologic factors and prognosis. The regulation of EGFR and IRAK1 by miR-146a was examined with miR-146a–transfected gastric cancer cells. Moreover, we analyzed the association between miR-146a levels and the G/C single nucleotide polymorphism (SNP) within pre-miR-146a seed sequences in 76 gastric cancer samples, using direct sequencing of genomic DNA. Results: In 90 clinical samples of gastric cancer, miR-146a levels in cancer tissues were significantly lower than those in the corresponding noncancerous tissue (P < 0.001). Lower levels of miR-146a were associated with lymph node metastasis and venous invasion (P < 0.05). Moreover, a lower level of miR-146a was an independent prognostic factor for overall survival (P = 0.003). Ectopic expression of miR-146a inhibited migration and invasion and downregulated EGFR and IRAK1 expression in gastric cancer cells. In addition, G/C SNP within the pre-miR-146a seed sequence significantly reduced miR-146a levels in the GG genotype compared with the CC genotype. Conclusions: MiR-146a contains an SNP, which is associated with mature miR-146a expression. MiR-146a targeting of EGFR and IRAK1 is an independent prognostic factor in gastric cancer cases. Clin Cancer Res; 17(13); 4277–84. ©2011 AACR.

Introduction: Esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy with extremely poor overall survival. There is no available molecular marker in clinical application until now. SATB1(special AT-rich binding protein 1) tether genomic DNA in nucleolus and hold three-dimensional structure of genomic DNA. We evaluated SATB1 expression using clinical samples from patients of ESCC and analyzed the association with clinicopathological factors and prognosis to identify novel prognostic marker in ESCC. In addition, we investigated the function of SATB1 using ESCC cells. We focused on matrix metalloproteinase protein (MMP3), which has a SATB1 binding site in its upstream. Materials and Methods: We accumulated 71 samples of ESCC patients who underwent primary tumor resection. The relative expression levels of SATB1 were obtained by normalizing the amount of SATB1 mRNA with respect to GAPDH mRNA as an endogenous control for each sample. We divided 71 ESCC samples into 2 groups, which are SATB1 low expression group (n=35) and SATB1 high expression group (n=36) according to the median of SATB1 expression level, and performed the comparison between two groups. In order to elucidate biological significance, we generated SATB1 overexpression stable transfectants in KYSE150, and performed migration and invasion assays. Next, we knockdowned SATB1 expression of KYSE410, which are ESCC lines, using SATB1 siRNA and evaluated the capability of migration and invasion. Moreover, to validate whether SATB1 altered MMP3 genomic locus, we analyzed comparative genomic hybridization (CGH) array obtained from 54 esophageal cancer samples. We examined MMP3 expression in SATB1 overexpression cells using qRT-PCR. Result: The SATB1 high expression group showed greater tumor depth than the low expression group (P=0.003). In the overall survival curve, patients in the SATB1 high expression group had a significant poorer prognosis than those in the SATB1 low expression group (P=0.013). Multivariate analysis showed that the level of SATB1 expression was an independent prognostic predictor (OR; 1.56, 95%CI; 1.04 - 2.44, P = 0.043). SATB1 transfectants significantly increased the capability of migration and invasion compared to mock (P Conclusion: We indicated that SATB1 is novel prognostic factor in ESCC and that SATB1 promote migration and invasion of ESCC cells. SATB1 may be a promising new candidate for targeted therapies for ESCC. Citation Format: Yusuke Tsuruda, Ryunosuke Kogo, Shuhei Ito, Akihiro Kitagawa, Atsushi Fujii, Shotaro Kuramitsu, Dai Simizu, Yosuke Kuroda, Hidetoshi Eguchi, Tkaaki Masuda, Shoji Natsugoe, Masaki Mori, Koshi Mimori. SATB1 is a novel prognostic factor in esophageal cancer patients and promotes migration and invasion of esophageal cancer cells [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 1552.

// Ryunosuke Kogo 1 , Christine How 1 , Naz Chaudary 1 , Jeff Bruce 1 , Wei Shi 1 , Richard P. Hill 1,2,3 , Payam Zahedi 1 , Kenneth W. Yip 1 and Fei-Fei Liu 1,2,3,4 1 Ontario Cancer Institute, University Health Network (UHN), Toronto, Ontario, Canada 2 Department of Medical Biophysics, University of Toronto, Toronto, Canada 3 Department of Radiation Oncology, University of Toronto, Toronto, Canada 4 Department of Radiation Oncology, Princess Margaret Cancer Centre, UHN, Toronto, Canada Correspondence: Fei-Fei Liu, email: // Keywords : miR-218, survivin, cervical cancer, migration, invasion Received : September 30, 2014 Accepted : November 24, 2014 Published : November 25, 2014 Abstract Cervical cancer is the third most common cancer in women worldwide. In the present study, global microRNA profiling for 79 cervical cancer patient samples led to the identification of miR-218 down-regulation in cervical cancer tissues compared to normal cervical tissues. Lower miR-218 expression was associated significantly with worse overall survival (OS), disease-free survival (DFS), and pelvic/aortic lymph node recurrence. In vitro , miR-218 over-expression decreased clonogenicity, migration, and invasion. Survivin (BIRC5) was subsequently identified as an important cervical cancer target of miR-218 using in silico prediction, mRNA profiling, and quantitative real-time PCR (qRT-PCR). Concordant with miR-218 over-expression, survivin knockdown by siRNA decreased clonogenicity, migration, and invasion. YM155, a small molecule survivin inhibitor, significantly suppressed tumor growth and lymph node metastasis in vivo . Our findings demonstrate that the miR-218~survivin axis inhibits cervical cancer progression by regulating clonogenicity, migration, and invasion, and suggest that the inhibition of survivin could be a potential therapeutic strategy to improve outcome in this disease.

Circulating tumor cells (CTC) in blood have attracted attention both as potential seeds for metastasis and as biomarkers. However, most CTC detection systems might miss epithelial–mesenchymal transition (EMT)-induced metastatic cells because detection is based on epithelial markers. First, to discover novel markers capable of detecting CTCs in which EMT has not been repressed, microarray analysis of 132 colorectal cancers (CRC) from Japanese patients was conducted, and 2,969 genes were detected that were overexpressed relative to normal colon mucosa. From the detected genes, we selected those that were overexpressed CRC with distant metastasis. Then, we analyzed the CRC metastasis-specific genes (n = 22) to determine whether they were expressed in normal circulation. As a result, PLS3 was discovered as a CTC marker that was expressed in metastatic CRC cells but not in normal circulation. Using fluorescent immunocytochemistry, we validated that PLS3 was expressed in EMT-induced CTC in peripheral blood from patients with CRC with distant metastasis. PLS3-expressing cells were detected in the peripheral blood of approximately one-third of an independent set of 711 Japanese patients with CRC. Multivariate analysis showed that PLS3-positive CTC was independently associated with prognosis in the training set (n = 381) and the validation set [n = 330; HR = 2.17; 95% confidence interval (CI) = 1.38–3.40 and HR = 3.92; 95% CI = 2.27–6.85]. The association between PLS3-positive CTC and prognosis was particularly strong in patients with Dukes B (HR = 4.07; 95% CI = 1.50–11.57) and Dukes C (HR = 2.57; 95% CI = 1.42–4.63). PLS3 is a novel marker for metastatic CRC cells, and it possesses significant prognostic value. Cancer Res; 73(7); 2059–69. ©2012 AACR.

The functions of many long non-coding RNAs (ncRNAs) in human cancers have not yet been elucidated. The long ncRNA HOTAIR is expressed from the developmental HOXC locus located on chromosome 12q13.13. Previous reports have demonstrated that HOTAIR associates with chromatin modifications in cooperation with the Polycomb complex PRC2, and promotes breast and colorectal cancer metastasis. In this study, we examined the clinical significance of HOTAIR expression in patients with hepatocellular carcinoma (HCC). HOTAIR expression was detected in primary HCCs in 13 out of 64 patients. Patients with HOTAIR expression had significantly poorer prognoses and a larger primary tumor size than those without HOTAIR expression, similar to studies in breast and colorectal cancers. Moreover, introduction of human HOTAIR into liver cancer cells revealed that HOTAIR promoted more rapid proliferation compared to control cells. Thus, although the clinical significance of HOTAIR expression in HCC may not be as pronounced as that in breast and colorectal cancers, the current study demonstrates that HOTAIR expression is associated with HCC progression, warranting further studies.

The mammalian target of rapamycin (mTOR) is a downstream integrator of essential pathways. mTOR signaling is frequently dysregulated in a variety of human cancers, and in silico analysis has revealed two miR-144 binding sites in the mTOR 3' untranslated region. We investigated the clinicopathologic magnitude of the mTOR pathway regulating microRNA, miR-144 in colorectal cancer (CRC) cases. The regulation of mTOR by miR-144 was examined with inhibitor miR-144-transfected cells. We also investigated changes in sensitivity to the mTOR inhibitor, rapamycin, in inhibitor miR-144-transfected cells. Quantitative RT-PCR was used to evaluate the clinicopathologic significance of miR-144 expression in 137 CRC. Furthermore, we assessed the correlation between CRC prognosis and the expression of 16 genes in the Akt/mTOR pathway. In vitro assays showed that mTOR is a direct target of miR-144, and downregulation of miR-144 facilitated proliferation of CRC cell line, HT29. In addition, the viability of HT29 cells with downregulated miR-144 expression was significantly reduced with rapamycin treatment. Low expression levels of miR-144 were associated with enhanced malignant potential such as venous invasion (P = 0.0013), liver metastasis (P = 0.08), liver recurrence (P = 0.0058) and poor prognosis (P = 0.0041). Multivariate analysis indicated that low miR-144 expression was an independent prognostic factor for survival. Among many genes consisting of the mTOR pathway, only high expression of Rictor was associated with poor prognosis of CRC. miR-144 is a meaningful prognostic marker. Downregulation of miR-144 leads to poor prognosis of CRC patients via activation of the mTOR signaling pathway.

Cell growth demands new protein synthesis, which requires nucleolar ribosomal functions. Ribosome biogenesis consumes a large proportion of the cell's resources and energy, and so is tightly regulated through an intricate signaling network to guarantee fidelity. Thus, events that impair ribosome biogenesis cause nucleolar stress. In response to this stress, several nucleolar ribosomal proteins (RPs) translocate to the nucleoplasm and bind to MDM2. MDM2‐mediated ubiquitination and degradation of the tumor suppressor p53 is then blocked, resulting in p53 accumulation and the induction of p53‐dependent cell cycle arrest and apoptosis. Nucleolar stress is therefore a quality control surveillance mechanism that monitors the synthesis and assembly of the rRNA and protein components of ribosomes. Although nucleolar stress signaling pathways have been extensively analyzed, critical questions remain about their regulatory mechanisms. For example, how do RPs translocate from the nucleolus to the nucleoplasm to exert their functions, and do these p53‐regulating RPs influence the prognosis of human cancer patients? Our laboratory recently identified the nucleolar protein PICT1 as a novel regulator of nucleolar stress. PICT1 sequesters the ribosomal protein RPL11 in the nucleolus, preventing it from binding to MDM2. MDM2 is then free to degrade p53, favoring tumor cell growth. Accordingly, the level of PICT1 in a tumor is becoming a useful prognostic marker for human cancers. This review summarizes the evidence that links nucleolar stress to tumorigenesis, and casts PICT1 as an oncogenic player in human cancer biology. (Cancer Sci 2012; 103: 632–637)

The functional impact of recently discovered long noncoding RNAs (ncRNAs) in human cancer remains to be clarified. One long ncRNA which has attracted attention is the Hox transcript antisense intergenic RNA termed HOTAIR, a long ncRNA expressed from the developmental HOXC locus located on chromosome 12q13.13. In cooperation with Polycomb complex PRC2, the HOTAIR long ncRNA is reported to reprogram chromatin organization and promote breast cancer metastasis. In this study, we examined the status and function of HOTAIR in patients with stage IV colorectal cancer (CRC) who have liver metastases and a poor prognosis. HOTAIR expression levels were higher in cancerous tissues than in corresponding noncancerous tissues and high HOTAIR expression correlated tightly with the presence of liver metastasis. Moreover, patients with high HOTAIR expression had a relatively poorer prognosis. In a subset of 32 CRC specimens, gene set enrichment analysis using cDNA array data revealed a close correlation between expression of HOTAIR and members of the PRC2 complex (SUZ12, EZH2, and H3K27me3). Our findings suggest that HOTAIR expression is associated with a genome-wide reprogramming of PRC2 function not only in breast cancer but also in CRC, where upregulation of this long ncRNA may be a critical element in metastatic progression. Cancer Res; 71(20); 6320–6. ©2011 AACR.

Cyclin D1 plays important roles in esophageal squamous cell carcinoma (ESCC) cases by amplification of the 11q13.3 locus. FBXO31 is a subunit of the SCF ubiquitin ligase, which targets cyclin D1 for degradation. In this study, we clarified the clinical significance of FBXO31 and characterized the association between cyclin D1 and FBXO31 in ESCC cases. Total RNA was extracted from tumor tissues obtained from 68 ESCC patients who underwent surgical resection. FBXO31 expression levels were determined by quantitative RT-PCR, and both FBXO31 and cyclin D1 protein expression and localization were evaluated by immunohistochemistry (IHC). Furthermore, using CGH and gene expression array data of another subset, we validated the association between cyclin D1 genomic amplification and FBXO31 expression levels. Higher FBXO31 expression levels significantly correlated with depth of tumor invasion and clinical stage (P

The aim of this study was to identify and evaluate novel prognostic markers for gastric cancer. Differential mRNA displays comparing paired tumor/normal stomach samples were assessed. Several differentially expressed cDNA fragments of candidate genes were identified, and one of these was further studied using quantitative reverse transcription-PCR in 140 human gastric carcinomas. To evaluate protein expression, immunohistochemical staining was performed in selected cases. One of the genes abundantly expressed in tumor tissue on the differential mRNA displays was identified as histone deacetylase 1 (HDAC). HDAC was overexpressed in the tumor tissue in 77% of the cases as determined by quantitative reverse transcription-PCR. Immunohistochemical staining revealed analogous results, showing strong expression in cancer cells. Patients were then classified into high (n=78) and low (n=62) expression groups according to the mean value of HDAC expression. High frequencies of lymph vessel and vascular vessel permeations, and advanced stage of the disease were recognized in the high expression group compared to the low expression group (p

MicroRNA (miR)-218 down-regulation has been reported in numerous human malignancies. In cervical cancer, we identified that lower miR-218 expression was significantly associated with poorer overall survival, disease-free survival, and pelvic/para-aortic lymph node recurrence. Further analyses of cervical cancer data from The Cancer Genome Atlas (TCGA) identified that this down-regulation was associated with a genomic locus loss (hsa-mir-218-1:4p15.31, hsa-mir-218-2:5q34, n=105). The objective of the current study was to elucidate the cellular and molecular functions of miR-218. MiR-218 transfection into cervical cancer cells (SiHa and ME-180) significantly reduced cell migration (by 66% and 89%, respectively), invasion (by 49% and 67%, respectively), and clonogenic capacity (by 42% and 53%, respectively), relative to control-transfected cells (P Citation Format: Ryunosuke Kogo, Christine How, Jeff Bruce, Willa Shi, Kenneth W. Yip, Laurie Ailles, Fei-Fei Liu. The microRNA-218-survivin axis regulates cervical cancer cell migration and invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4369. doi:10.1158/1538-7445.AM2014-4369

INTRODUCTION MicroRNAs (miRs) have been implicated in various human malignancies and are being developed as diagnostic, predictive, and prognostic biomarkers. Our previous global profiling studies found miR-449a to be over-expressed in archival lymph node-negative invasive ductal breast carcinoma samples (n=74) when compared to normal tissues. Furthermore, miR-449a expression level was significantly associated with relapse. The mechanism(s) by which miR-449a functions remains unknown, and the aim of the current study was therefore to elucidate the roles and mRNA targets of miR-449a in breast cancer. MATERIAL AND METHODS MiR-449a expression was evaluated in six human breast cancer cell lines (T47D, MDA-MB-468, MDA-MB-231, MCF-7, MDA-MB-453, and ZR75-1). Three of these cell lines, T47D, MMDA-MB-468, and MDA-MB-231, were selected and used to assess the biological effects of miR-449a on cell viability, clonogenicity, cell migration, and invasion. Downstream target genes were identified by combining in silico miRWalk prediction and cell line/patient sample microarray data. Targets were validated using qRT-PCR and luciferase reporter assays. RESULTS Consistent with our global profiling work, several (4/6) of the tested breast cancer cell lines over-expressed miR-449a. CDC20B, the miR-449a host gene and an essential regulator of cell division, was also over-expressed. Knockdown of miR-449a resulted in significantly decreased cell viability, clonogenic survival, migration, and invasion. MiR-449a candidate targets were then identified by integrating in silico prediction algorithms, cell line mRNA expression profiling (Affymetrix Human Genome U133 Plus 2.0), and clinical specimen mRNA expression data. Two genes, CRIP2 (Cysteine rich protein 2, a transcription factor) and PRKAG1 (Protein Kinase, AMP-Activated, Gamma 1 Non-Catalytic), were verified as miR-449a targets using qRT-PCR. Moreover, direct interactions between miR-449a and the 3′-UTRs of both CRIP2 and PRKAG1 were confirmed using luciferase reporter assays. Inhibition of CRIP2 by miR-449a allowed for NF-κB-induced transcription of survival, proliferation, and growth-related genes, providing further evidence for the role of miR-449a in breast cancer progression. CONCLUSIONS MiR-449a is over-expressed in breast cancer cells, promoting cellular proliferation, migration, and invasion. One mechanism by which miR-449a functions is to down-regulate CRIP2, leading to NF-kB activation and possible cancer progression. Further investigations into gene regulation by miR-449a may reveal other promising targets for the management of breast cancer. Citation Format: Wei Shi, Matthew Lee, Ryunosuke Kogo, Jeff Bruce, Christine How, Kenneth W. Yip, Fei-Fei Liu. MiR-449a promotes breast cancer progression by activating the NF-κB pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4372. doi:10.1158/1538-7445.AM2014-4372

The tumor-suppressive MDM2-p53 intracellular signaling pathway is activated by cellular stress. Such stress includes DNA damage, which triggers the ATM-Chk2 and ATR-Chk1 cascades; infection or oncogene activation, which induce the p19ARF pathway; and nucleolar stress, which initiates a cascade mediated by ribosomal proteins (RPs), particularly RPL5, RPL11, RPL23, and RPS7. These RPs are usually located in the nucleolus but are rapidly released into the nucleoplasm upon nucleolar stress, activating the MDM2-p53 pathway. However, it is unknown whether there are direct connections between p53-activating RP genes and cancer development in vivo, and which mechanism these RPs employ to translocate from the nucleolus to the nucleoplasm in response to stress. PICT1/GLTSCR2 was originally identified as a candidate tumor suppressor gene located at human chromosome 19q13.32, the site of deletions in human gliomas. Subsequent in vitro studies have demonstrated that PICT1 regulates the stability of PTEN, and that low PICT1 expression caused growth advantage. Based on these findings, PICT1 has been deemed a tumor suppressor. Other evidence, however, suggests that PICT1 may not always dampen cancer progression. In oligodendroglial tumors, loss of heterozygosity (LOH) at chromosome 19q13 is associated with longer disease-free survival after chemotherapy. These contradictory observations indicate that one or more genes mapped to chromosome 19q may contribute to brain cancer development. We speculated that PICT1 might be an important chromosome 19q–mapped gene that regulates tumor progression. To clarify PICT1 function, we generated PICT1-deficient mice and ES cells and carried out extensive biochemical and molecular analyses. Our results show that PICT1 is a nucleolar protein essential for murine embryogenesis and ES cell survival. Even without DNA damage, PICT1 loss results in p53-dependent G1 arrest and apoptosis in vitro and in vivo. Strikingly, PICT1-deficient cells accumulate p53 due to impaired MDM2 function. We further show that PICT1 binds to RPL11, and that RPL11 is released from nucleoli in the absence of PICT1. In PICT1-deficient cells, increased binding of RPL11 to MDM2 blocks MDM2-mediated ubiquitination of p53, leading to p53 accumulation and repression of cell proliferation. In cases of human colon and esophageal cancer, patients with low PICT1 expression have better prognoses. Similarly, when shRNA is used to deplete PICT1 in various tumor cell lines with intact p53 signaling, the cells grow more slowly and accumulate p53. These data suggest that PICT1 is a novel key regulator of the MDM2-p53 pathway and promotes tumor progression by retaining RPL11 in the nucleolus. Thus, PICT1 is an important cancer-related gene that, at the very least, does not behave like a typical tumor suppressor and may instead have oncogenic effects. Studies on identification of factors which influence PICT1 expression or stability, or interfering with PICT1-RPL11 binding may lead to new cancer therapeutics, especially for individuals with intact-p53 signaling. This abstract is also presented as Poster B30. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR9.