High Metastatic Ability Research Articles

Bioinformatics-based analysis reveals elevated MFSD12 as a key promoter of cell proliferation and a potential therapeutic target in melanoma

Although recent therapeutic advances based on our understanding of molecular phenomena have prolonged the survival of melanoma patients, the prognosis of melanoma remains dismal and further understanding of the underlying mechanism of melanoma progression is needed. In this study, differential expression analyses revealed that many genes, including AKT1 and CDK2, play important roles in melanoma. Functional analyses of differentially expressed genes (DEGs), obtained from the GEO (Gene Expression Omnibus) database, indicated that high proliferative and metastatic abilities are the main characteristics of melanoma and that the PI3K and MAPK pathways play essential roles in melanoma progression. Among these DEGs, major facilitator superfamily domain-containing 12 (MFSD12) was found to have significantly and specifically upregulated expression in melanoma, and elevated MFSD12 level promoted cell proliferation by promoting cell cycle progression. Mechanistically, MFSD12 upregulation was found to activate PI3K signaling, and a PI3K inhibitor reversed the increase in cell proliferation endowed by MFSD12 upregulation. Clinically, high MFSD12 expression was positively associated with shorter overall survival (OS) and disease-free survival (DFS) in melanoma patients, and MFSD12 was an independent prognostic factor for OS and DFS in melanoma patients. Therapeutically, in vivo assays further confirmed that MFSD12 interference inhibited tumor growth and lung metastasis in melanoma. In conclusion, elevated MFSD12 expression promotes melanoma cell proliferation, and MFSD12 is a valuable prognostic biomarker and promising therapeutic target in melanoma.

Odd-skipped related transcription factor 1 (OSR1) suppresses tongue squamous cell carcinoma migration and invasion through inhibiting NF-κB pathway

Tongue squamous cell carcinoma (TSCC) is the most common cancers of oral, owing to the high invasive and metastatic ability, patients with TSCC have poor prognosis, it's important to explore the regulatory mechanism of TSCC invasion and metastasis. Previous studies suggest OSR1 suppresses the progression of gastric cancer and renal cell carcinoma, but its role in TSCC hasn’t been studied. Here, we found OSR1 was downregulated in TSCC cells and specimens, Transwell and 3D spheroid invasion assay suggested OSR1 overexpression inhibited TSCC cell migration and invasion, while its knockdown promoted TSCC cell migration and invasion. Mechanism analysis found OSR1 expression was negatively correlated with NF-κB pathway and its targets. Western blot and NF-κB activity analysis suggested OSR1 inhibited NF-κB activity. Double inhibition of OSR1 and NF-κB significantly inhibited TSCC cell migration and invasion. These findings suggested OSR1 inhibited TSCC cell migration and invasion through inhibiting NF-κB pathway.

MDM2 Overexpression Modulates the Angiogenesis-Related Gene Expression Profile of Prostate Cancer Cells.

The Murine Double Minute 2 (MDM2) amplification or overexpression has been found in many tumors with high metastatic and angiogenic ability. Our experiments were designed to explore the impact of MDM2 overexpression, specifically on the levels of angiogenesis-related genes, which can also play a major role in tumor propagation and increase its metastatic potential. In the present study, we have used the human angiogenesis RT2 profiler PCR array to compare the gene expression profile between LNCaP and LNCaP-MST (MDM2 transfected) prostate cancer cells, along with LNCaP-MST cells treated with Nutlin-3, an MDM2 specific inhibitor. As a result of the overexpression of MDM2 gene in LNCaP-MST (10.3-fold), Thrombospondin 1 (THBS1), Tumor necrosis factor alpha (TNF-α) and Matrix metallopeptidase 9 (MMP9) were also found to be significantly up-regulated while genes such as Epiregulin (EREG), Tissue inhibitor of metalloproteinases 1 (TIMP1) were down-regulated. Also, we determined the total MMP activity and MMP9 expression in LNCaP, LNCaP-MST and SJSA-1 cells. Our results indicated that MDM2 level is positively correlated with MMP activity and MMP9 secretion. Our findings offer strong supporting evidence that MDM2 can impact growth and metastatic potential of cancer cells through tilting the balance towards pro-angiogenic mechanisms.

Alveolar soft part sarcoma: Clinical presentation, treatment, and outcome in a series of 13 patients

BackgroundAlveolar soft part sarcoma (ASPS) is a rare soft tissue tumor that typically affects young patients. Similar to other soft tissue sarcomas, it has high pulmonary metastasis ability, whereas compared with other soft tissue sarcomas, it has high brain metastasis ability. Because of the rarity of the disease, most studies on ASPS have been case reports and small series studies. MethodWe performed a retrospective study to evaluate the clinical and pathological features and oncological results in a consecutive series of patients with localized or metastatic ASPS treated at our institute between 1994 and 2014. Demographics, location, severity of disease, treatment provided, progression-free survival, and overall survival were evaluated. ResultsA total of 13 patients were investigated. The most common locations of primary tumor were the thigh (n = 6, 47%), followed by the flank (n = 3, 23%), forearm (n = 2, 15%), and calf (n = 2, 15%). Three patients were initially diagnosed as having hemangiomas elsewhere. These patients received unplanned intralesional excision. All the patients received wide tumor resection at our institute. Over the average follow-up period of 80.5 months (range: 36–133 months), the 5-year overall survival rate was 67.5%. Four patients were continuously disease free (31%), six were living with disease (46%), and three died of disease (23%). Of nine patients who presented with distant pulmonary metastasis, two had bony and brain metastases. The 5-year survival rate was 66.7% in patients who received chemotherapy and those who did not (p = 0.941). ConclusionThe treatment strategy for ASPS is wide resection, and postoperative chemotherapy may be crucial for long-term survival. In addition, this type of tumor has a high distant metastasis rate at the time of diagnosis, particularly in the lungs and brain.

FYN promotes mesenchymal phenotypes of basal type breast cancer cells through STAT5/NOTCH2 signaling node.

Basal type breast cancer is the most aggressive and has mesenchymal features with a high metastatic ability. However, the signaling node that determines the basal type features in breast cancer remains obscure. Here, we report that FYN among SRC family kinases is required for the maintenance of basal type breast cancer subtype. Importantly, FYN enhanced NOTCH2 activation in basal type breast cancer cells through STAT5-mediated upregulation of Jagged-1 and DLL4 NOTCH ligands, thereby contributed to mesenchymal phenotypes. In addition, we found that high levels of FYN persist in basal type breast cancer cells by a positive feedback loop between FYN and STAT5. FYN interacted directly with STAT5 and increased p-STAT5 that further acts as a transcription factor for FYN. Taken together, our findings demonstrate a pivotal role of FYN and its downstream effectors in maintaining the basal type features in breast cancer.

Inhibition of Histone Deacetylases Reverses Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells through a Slug Mediated Mechanism

High metastatic ability and poor clinical outcome are the most known clinical features of the triple-negative breast tumors. Given that the tumor cells undergoing epithelial-mesenchymal transition (EMT) often gain malignant and invasive features, we have investigated the possibility of EMT reversal in triple-negative breast cancer cells by targeting the epigenetic-modifying enzymatic complexes named histone deacetylases (HDACs) and examined the possible mechanism underlying the HDACs-based inversion in model MDA-MB-231 cells. Cells were treated with a maximal tolerable 200 nM concentrations of classical HDACs inhibitor Trichostatin A (TSA) for 48 h and afterwards the invasiveness and immigration of the cells were evaluated in TransWell Invasion Scratch Wound Healing assays. Then, in treated and control cells, quantitative real time-PCR reacions were performed for assessing the gene expression of EMT biomarkers E-cadherin, Vimentin and transcriptional factor Slug. After TSA treatment, the invasion and migration properties MDA-MB-231 cells significantly decreased, gene expression of E-cadherin was significantly up-regulated, while the levels of Slug and Vimentin encoding mRNAs were suppressed. We conclude that inhibition of HDACs in triple-negative breast cancer cells may lead to inversion of EMT and the decrease of invasiveness by down-regulating the gene expression of Slug. Since EMT is known as a pre-metastatic process, triple-negative breast tumors, the EMT reversal effects of HDACs inhibition may reduce tumor cell metastasis.

Deubiquitinase USP9X promotes cell migration, invasion and inhibits apoptosis of human pancreatic cancer.

Ubiquitin specific peptidase9, X-linked (USP9X), a significant regulatory protease in protein ubiquitination, has been proven to act as a proto-oncogene in several types of cancers, such as cervix, colon, breast, brain and lung cancers. The prognosis of patients with pancreatic ductal adenocarcinoma (PDAC) is extremely poor due to its high invasive and metastatic abilities. Nevertheless, whether USP9X acts as a proto-oncogene or a tumor-suppressor gene in PDAC is controversial and the mechanism of metastasis remains unknown. The present study focused on the effect of USP9X on the migration, invasion and apoptosis of PADC cells. We analyzed the expression of USP9X in pancreatic cancer tissues of different pathologic grades by immunohistochemical analysis. USP9X expression in the pancreatic cancer tissues was markedly increased in contrast to that noted in the adjacent non-tumor tissues. USP9X expression in PANC-1 cells was downregulated after transfection of shRNA-USP9X. Knockdown of endogenous USP9X expression evidently inhibited the migration and invasion of PANC-1 cells, and promoted cell apoptosis. Meanwhile, expression levels of Snail, Twist, N-cadherin and vimentin were downregulated. E-cadherin expression was negatively correlated with USP9X expression and the expression of survivin was also downregulated in the PANC-1 cells. In brief, USP9X promoted the migration and invasion of PANC-1 cells probably by provoking epithelial-mesenchymal transition, and also inhibited apoptosis. We believe that USP9X is a major oncogene that may play a significant role in the treatment and prognosis of PDAC.

BLIMP1 Induces Transient Metastatic Heterogeneity in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic and deadly cancers. Despite the clinical significance of metastatic spread, our understanding of molecular mechanisms that drive PDAC metastatic ability remains limited. By generating a genetically engineered mouse model of human PDAC, we uncover a transient subpopulation of cancer cells with exceptionally high metastatic ability. Global gene expression profiling and functional analyses uncovered the transcription factor BLIMP1 as a driver of PDAC metastasis. The highly metastatic PDAC subpopulation is enriched for hypoxia-induced genes, and hypoxia-mediated induction of BLIMP1 contributes to the regulation of a subset of hypoxia-associated gene expression programs. These findings support a model in which upregulation of BLIMP1 links microenvironmental cues to a metastatic stem cell character.Significance: PDAC is an almost uniformly lethal cancer, largely due to its tendency for metastasis. We define a highly metastatic subpopulation of cancer cells, uncover a key transcriptional regulator of metastatic ability, and define hypoxia as an important factor within the tumor microenvironment that increases metastatic proclivity. Cancer Discov; 7(10); 1184-99. ©2017 AACR.See related commentary by Vakoc and Tuveson, p. 1067This article is highlighted in the In This Issue feature, p. 1047.

Sphingosine-1-phosphate suppresses chondrosarcoma metastasis by upregulation of tissue inhibitor of metalloproteinase 3 through suppressing miR-101 expression.

Chondrosarcoma is the second most common primary malignancy form of bone cancer, exhibiting resistance to chemotherapy and radiation therapy as well as developing high metastasis ability in late‐stage tumors. Thus, understanding the metastatic processes of chondrosarcoma is considered a strategy for the treatment of this disease. Sphingosine 1‐phosphate (S1P), a bioactive sphingolipid, is produced intracellularly by sphingosine kinase (SphK) and is regarded as a second signaling molecule that regulates inflammation, proliferation, angiogenesis, and metastasis. However, the effect of S1P on chondrosarcoma remains uncertain. As demonstrated by the transwell, immunoblotting, and real‐time PCR analyses, we found that S1P inhibited cell migration and MMP‐2 expression through the upregulation of the tissue inhibitor of metalloproteinase‐3 (TIMP‐3) expression in human chondrosarcoma cells. Additionally, we also showed that microRNA (miRNA)‐101, which targets the 3′ untranslated region (3′UTR) of TIMP‐3, decreased significantly following S1P treatment. After transfection with miR‐101 mimics, the S1P‐regulated cell migration and TIMP‐3 expression were both reversed. Furthermore, we also showed that the S1P‐inhibited cell migration is mediated through the c‐Src/MEK/ERK signaling axis. Meanwhile, the in vivo study indicated that overexpression of SphK1 decreases chondrosarcoma metastasis to the lungs. Our results illustrate the clinical significance between SphK1, TIMP‐3, and miR‐101 in human chondrosarcoma patients. Taken together, our results suggest that S1P and miR‐101 may prove to be potential therapeutic targets for future chondrosarcoma treatment.

Peri-tumor associated fibroblasts promote intrahepatic metastasis of hepatocellular carcinoma by recruiting cancer stem cells

Fibroblasts have been reported to play an important role in hepatocellular carcinoma (HCC). However, the role of fibroblasts have not been fully understood. Conditioned medium collected from human peri-tumor tissue-derived fibroblasts (CM-pTAFs) showed high metastasis ability than human HCC tissues-derived fibroblasts (CM-TAFs). To determine what component was secreted from fibroblasts, we used Bio-Plex analysis system and compared the factors secreted from CM-pTAFs and CM-TAFs, found a series of up-regulated cytokines in the CM-pTAFs, including IL-6, CCL2, CXCL1, CXCL8, SCGF-β, HGF and VEGF. Pretreatment of IL-6 inhibitor Tocilizumab could inhibit metastasis the HCC cell treated with CM-pTAFs in vitro and in vivo. The expression of CCR2 and CXCR1 were up-regulated after CM-pTAFs treatment in HCC cell line SMMC-7721. Flow cytometric analysis experiment showed that most CCR2 or CXCR1 positive cells were also EpCAM positive. In vitro studies also showed that CM-pTAFs could increase stemness of SMMC-7721. In addition, neutralization of SCGF-β and HGF could significantly reduce metastasis and viability of cancer stem cells treated with CM-pTAFs. Taken together, these results indicated that the peri-tumor tissues derived fibroblasts may promote development of HCC by recruiting cancer stem cells and maintaining their stemness characteristic.

Abstract 3517: FOXC1 promotes pancreatic cancer cell proliferation and metastasis

Abstract BACKGROUD: Pancreatic cancer is one of the most aggressive cancers due to ineffective treatment options and its high metastatic abilities. This highlights the significance for the development of effective modalities to treat this deadly disease. Dysregulation of IGF-1R signaling has been shown to be involved in the invasive and metastatic potential of several cancer types. Currently, the mechanism by which IGF-1R promotes pancreatic cancer is not well understood. Our preliminary transcription factor profiling array indicated that FOXC1 was regulated by IGF-1R. Therefore, in the present study, we investigated the role of FOXC1 in pancreatic carcinogenesis. METHODS: Western blot and qRT-PCR analysis were done to measure the expression levels of FOXC1 in a panel of pancreatic cancer cell lines along with normal pancreatic cells. IHC analysis of the levels of FOXC1 by tumor stage in human pancreatic ductal adenocarcinoma tissues and normal pancreas tissue was performed using tissue microarray. Immunofluorescence analysis was performed to compare the expression levels of FOXC1 in normal and pancreatic cancer cells. To investigate the role of FOXC1 on pancreatic cancer cell growth and metastasis, we used an RNA interference approach to silence FOXC1 in HPAC pancreatic cancer cells. MTS assay was performed to study the cell viability. Metastatic capabilities of FOXC1 silenced HPAC cells were examined using matrigel invasion, scratch assay, and soft agar colony forming assays. To further confirm, Western blotting analysis was done to evaluate the key intermediates involved in proliferation, epithelial mesenchymal transition, and apoptosis. Exogenous expression of FOXC1 in MIAPaCa-2 cells was also used to understand the oncogenic role of FOXC1 in pancreatic cancer. To further validate, xenograft studies with FOXC1 silenced HPAC cells were performed. RESULTS: Expression of FOXC1 was upregulated in pancreatic cancer cell lines. Silencing FOXC1 in HPAC cells drastically decreased the cell viability; however, it did not alter the viability of normal pancreatic cells. Furthermore, it also significantly reduced the invasive, migratory, and colony forming abilities of pancreatic cancer cells. Targeting FOXC1 significantly altered the key signaling molecules involved in proliferation, EMT, and apoptosis of HPAC cells. Exogenous overexpression of FOXC1 in MIAPaCa-2 cells also validated the oncogenic role of FOXC1. HPAC xenograft models further demonstrated the therapeutic role of FOXC1 in pancreatic cancer. CONCLUSION: To conclude, our results suggest that silencing FOXC1 effectively targets pancreatic cancer cells suggesting that it could be a potential novel therapeutic option. Citation Format: Ramadevi Subramani, Fernando A. Camacho, Carly Levin, Joshua Medel, Sai Navana Kolli, Sushmita Bose Nandy, Diego A. Pedroza, Rajkumar Lakshmanaswamy. FOXC1 promotes pancreatic cancer cell proliferation and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3517. doi:10.1158/1538-7445.AM2017-3517

Abstract 2985: Interaction between the malignant melanoma cell of the primary lesion and the metastatic lesion

Abstract Introduction: The prognosis of patients with malignant melanoma is extremely poor because of the frequent metastasis. We have occasionally experienced the clinical cases that malignant melanoma patients rapidly developed distant metastases after the surgical resection of primary cancer lesion. These cases suggested that a primary tumor may control a metastatic tumor. We hypothesized that primary melanoma cells might produce a factor(s) which regulate the progression of metastatic melanoma cells. Then we investigated the growth and invasion interaction between primary malignant melanoma cells and high metastatic melanoma cells. Materials and methods: We used two malignant melanoma cell lines, B16 as a parent primary melanoma cell line and a selected daughter cell line B16/BL6 with high metastatic ability. We investigated the invasive interaction between the parent B16 cells and daughter B16/BL6 cells in vitro. The conditioned medium from B16 cells and B16/BL6 cells was added to B16 cells or B16/BL6 cells. Then we examined the effect of these conditioned medium on the motility and proliferation of melanoma cells by MTT assay or invasion assay at 24 hours and 72 hours after the addiction of the conditioned medium. Results:The conditioned medium from B16 cells significantly (p=0.02) suppressed the invasion ability of B16/BL6 cells at 24 hours after the addiction of the conditioned medium. In contrast, the invasion ability of B16 cells were not affected by the conditioned medium from B16/BL6 cells at 24 hours. Each conditioned medium did not affect on the proliferation of both melanoma cell lines. On the other hand, at 72 hours after the addiction of the conditioned medium from B16 cells significantly (p=0.000003) suppressed the growth of B16/BL6 cells. Conclusion: Primary melanoma cells might down-regulate the progression of metastatic melanoma cells by a soluble factor(s). Citation Format: Takaharu Hatano, HIsashi Motomura, Heishiro Fujikawa, Masakazu Yashiro, Kishu Kitayama. Interaction between the malignant melanoma cell of the primary lesion and the metastatic lesion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2985. doi:10.1158/1538-7445.AM2017-2985

Neuron-specific enolase, histopathological types, and age as risk factors for bone metastases in lung cancer.

Lung cancer is a malignant tumor with high metastatic ability and bone is the most common site of distant metastasis of it. However, the independent risk factors for bone metastases of lung cancer remain largely to be elucidated. Here, we conducted a retrospective study to evaluate the correlation between clinical-pathological parameters, serum levels of neuron-specific enolase and CYFRA21-1, and bone metastases in lung cancer patients. The results revealed that patients with bone metastases were younger than those without metastases. Adenocarcinoma was the most frequent type of histopathology in patients with bone metastases. And the incidence of bone metastasis in patients with adenocarcinoma was significantly higher than those with other histopathological subtypes ( p < 0.001). Furthermore, the serum concentration of neuron-specific enolase was significantly higher in patients with bone lesions than those without bone metastases. Multivariate logistic regression analysis showed that patients' age (odds ratio = 1.024, p < 0.001), concentrations of neuron-specific enolase (odds ratio = 1.212, p = 0.004), and histopathological types (odds ratio = 0.995, p = 0.001) were the independent risk factors for bone metastases in patients with lung cancer. Thus, physicians should pay attention to these factors in order to identify bone metastasis earlier while patient was primarily diagnosed as having lung cancer.

Abstract 3484: A long non-coding RNA, H19, as a novel therapeutic target for pancreatic cancer metastasis

Abstract Long non-coding RNAs (lncRNAs) comprise mRNA-like transcripts longer than 200 ribonucleotides and lack significant open reading frames. Recently, long non-coding RNAs have been reported to play important roles in epigenetic regulation, and acts on precursors or inhibitor of micro RNA. In our previous studies, we injected PANC-1, human pancreatic cancer cells into the spleens of NOG mice, and established a lung metastasis-derived cell line from lung metastatic nodules. Lung metastasis derived pancreatic cells had a high metastatic ability to liver and lungs compared with the parental cells (Matsuda et al., Am J Pathol, 2014). To examine the different gene expressions between the lung metastasis derived cells and parental cells, we performed a DNA microarray analysis. We found that 11 genes were expressed at greater than 10-fold levels in lung metastasis derived cancer cells compared to their parental cells. H19 showed an 82.4-fold increase in expression levels, and it was the second most increased and the only non-protein coding gene in the 11 genes. H19 is an imprinted lncRNA transcribed exclusively from the maternal allele and H19 gene produces a 2.3 kilo base pair long non-coding RNA. Two microRNAs, miR-675-3p and miR-675-5p are embedded in the exon1 of H19 and these miRNAs are reported to correlate with cell proliferation. H19 is an oncofetal RNA expressed in developing embryos and in tumors including bladder, breast, gastric, pancreatic, hepatocellular, and prostate cancers. Studies have shown that H19 enhances cancer invasion and metastasis; however, its role in cancer remains debatable. We examined the roles of H19 in pancreatic cancer growth and metastasis. Quantitative RT-PCR analysis using human pancreatic cancer tissues showed that H19 was more highly expressed in cancer tissues than the adjacent normal tissues in 11 of 18 cases. Branched DNA in situ hybridization analysis using tissue microarrays showed that H19 was detected in 17% (23/139) of invasive ductal carcinomas, and its expression positively correlated with higher histological tumor grades (P &amp;lt; 0.0001). H19 was expressed in 9 pancreatic cancer cell lines at various levels. Overexpression of H19 in PANC-1 cells induced higher motilities, whereas H19 inhibition using shRNA and siRNA showed opposite results; however, cell growth rates were unaffected in vitro. Expression levels of miR-675-3p and miR-675-5p altered as similar to the H19 levels in both H19 increased PANC-1 cells and H19 decreased cells. Tail vein injection of H19 shRNA vector-transfected PANC-1 cells markedly inhibited metastasis in the liver and lungs of immunodeficient mice. These findings suggest that H19 plays an important role in pancreatic cancer metastasis and H19 is a novel therapeutic target for metastasis of pancreatic cancer. Citation Format: Toshiyuki Ishiwata, Hisashi Yoshimura, Yoko Matsuda, Naoshi Ishikawa, Kaiyo Takubo, Tomio Arai, Junko Aida. A long non-coding RNA, H19, as a novel therapeutic target for pancreatic cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3484. doi:10.1158/1538-7445.AM2017-3484

MiR-23a promotes TGF-β1-induced EMT and tumor metastasis in breast cancer cells by directly targeting CDH1 and activating Wnt/β-catenin signaling.

TGF-β1-induced epithelial-mesenchymal transition (EMT) has been proved to be associated with metastasis of breast cancer cells. We attempted to detect a novel mechanism that microRNAs mediated the TGF-β1-induced EMT in the process of breast cancer metastasis. Here we reported that the expression of miR-23a was higher in breast cancer cells with high metastasis ability and patients with lymph node metastasis and the treatment of TGF-β1 significantly upregulated the expression of miR-23a in breast cancer cells. We found that miR-23a was upregulated by TGF-β1 post-transcriptionally and Smads directly bound the RNA Smad binding element (R-SBE) of miR-23a. Functional studies showed that inhibition of miR-23a suppressed the TGF-β1-induced EMT, migration, invasion and metastasis of breast cancer both in vitro and in vivo. In addition, we determined that miR-23a directly targeted and suppressed CDH1, one important gene in EMT phenomenon. Notably, Wnt/β-catenin signaling was activated by the suppression of CDH1 in the miR-23a mediated process of TGF-β1-induced EMT and tumor invasion. These results demonstrate that miR-23a promotes TGF-β1-induced tumor metastasis in breast cancer by targeting CDH1 and activating Wnt/β-catenin signaling. Taken together, our results indicate a novel regulatory mechanism of TGF-β1-induced EMT and suggest that miR-23a might be a potential target in breast cancer therapy.

MiR‐105/93‐3p promotes metastasis, chemoresistance and stemness in TNBC and circulating miR‐105/93‐3p act as predictive biomarker for TNBC chemoresistance

Triple negative breast cancer (TNBC) is defined as lack of ER, PR and HER2 receptor and accounts for 15–20% of all breast cancers. The treatment for TNBC is a big challenge due to lack of hormone and target therapy and neoadjuvant chemotherapy only can achieve 20%–30% pathological complete response in TNBC patients. In the current study, we identified four highly expressing oncomiRs, miR‐301b, miR‐181a‐3p, miR‐105 and miR‐93‐3p that were significantly associated with poor survival in TNBC patients. To explore the oncogenic roles of four oncomiRs described above, these miRNAs were ectopically expressed in MB‐231/HCC70 cells and silenced in HCC1937/BT‐549 cells, respectively, for examining proliferation, metastasis, chemoresistance and stemness. MiR‐105/93‐3p overexpressing cells showed Cisplatin resistance and high metastatic ability whereas silence of miR‐105/93‐3p can decrease Cisplatin resistance, metastatic ability and stemness. Bioinformatics analysis showed that Wnt/b‐catenin is one of the most potential miR‐105/93‐3p downstream pathways. Ectopic overexpression of miR‐105 or miR‐93‐3p actives Wnt/b‐catenin signaling by TOP/FOP reporter assay. Bioinformatics, RNA‐IP and immunoblotting showed that SFRP1 is directly targeted and down‐regulated by miR‐105 and miR‐93‐3p. In addition, restoration of SFRP1 can overcome miR‐105/93‐3p mediated cisplatin resistance. High expression of miR‐105/93‐3p and low expression of SFRP1 strongly associated with TNBC poor survival even after chemotherapy treatment. The expression of miR‐105 and miR‐93‐3p inversely correlate with SFPR1 protein level in TNBC N‐T pair. Notably, circulating miR‐105 and miR‐93‐3p significantly higher expression in TNBC than non‐TNBC patients' serum and normal serum. Collectively, our data conducted that miR‐105/93‐3p activate Wnt/b‐catenin signaling through down‐regulate SFRP1 to promote TNBC poor survival and circulating miR‐105/93‐3p may act as predictive biomarker for TNBC poor survival.Support or Funding InformationMOST‐105‐2320‐B‐006‐054‐

Abstract P5-07-05: BRCA1-IRIS overexpression promotes and maintains the tumor initiating phenotype in TNBC cells: Implications for breast cancer early lesions

Abstract Introduction. Tumor-initiating (TIC) phenotype is potent inducer of dissemination of cancer cells from early lesions showing self-renewal, multi-lineage differentiation, chemo-resistance, and are defined as CD44+/CD24-/ALDHA+. Here, we demonstrate that BRCA1-IRIS overexpression (IRISOE) promotes TNBC formation by enhancing basal, EMT and TIC phenotype. In regards to TIC phenotype, we show that IRISOE elevates CD44 expression and ALDHA activity while prevents CD24 surface presentation. We present a novel pathway for CD24 internalization and confirm these data in animal model and tumor samples. Finally, we evaluated in pre-clinical models the ability of IRISOE to promote dissemination from early lesion and potential utility of IRIS inhibition to deplete TICs in TNBCs. Methods. We used western blot, qRT/PCR, immunofluorescence (IF) and IHC to establish association of IRISOE with basal, EMT and TIC phenotype. FACS analysis to determine IRISOE effect on the TIC phenotype. 2D and 3D culture to evaluate the utility of IRIS inhibition + a ligating anti-CD24 antibody on TICs. In vivo mice models to study early dissemination, metastasis formation and the effect of IRIS peptide on TIC tumor growth. Finally, patient cohort to correlate IRISOE to BRCA1 expression, stem and dissemination biomarkers in breast cancer patients. Results. We demonstrate an association between IRISOE and high basal and EMT phenotype. IRISOE leads to significant increase in basal biomarkers (EGFR, CK5, CK17, CDH3) and EMT markers (N-cadherin, vimentin, fibronectin, FOXC2, twist and snailI). Within a cohort of breast cancer patients (n=326), including subcohort of TNBC patients (n=72), IRISOE was associated with loss of BRCA1. Association between IRISOE and increase in TIC phenotype, in vitro and in vivo was documented. We propose EGFR-c-Src-cortactin signaling pathway induced by IRISOE to be involved in CD24 internalization and thus TIC phenotype. In support of that both western blot and IF analysis showed that while IRIS silencing in TNBC cells led to complete re-direction of cytoplasmic CD24 to the membrane, silencing of members of this pathway led to partial effect. Conversely, their overexpression also partially reversed the effect of IRIS silencing on the TIC phenotype. IRISOE could also induce tumorigenesis and metastasis in non-tumorigenic luminal A, MCF7 cells by enhancing TIC phenotype in them. Not only that, mice injected with low numbers of MCF7/IRISOE cells generate larger tumors, most likely, due to enhanced interactions with the stroma, an effect most likely not possible when large number of cells is injected instead. These tumors also showed high dissemination in the form of increased CTCs and DTCs in the blood and bone marrow, respectively. Inhibiting IRIS with specific peptide led to 80% regression of tumors developed using TICs in NSG mice and the effect was even more dramatic on the Sox2+-population remained within these tumors. Conclusion. This study reveals that IRISOE cells acquire TIC, basal and EMT phenotypes along with lack of BRCA1 expression and possess high dissemination and metastasis ability. Our results support inhibiting IRIS in TNBC patients to prevent early dissemination and metastasis. Citation Format: Sinha A, Paul BT, ElShamy WM. BRCA1-IRIS overexpression promotes and maintains the tumor initiating phenotype in TNBC cells: Implications for breast cancer early lesions [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-07-05.

Phosphorylation of cofilin-1 by ERK confers HDAC inhibitor resistance in hepatocellular carcinoma cells via decreased ROS-mediated mitochondria injury

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Despite the availability of several treatment strategies, resistance to chemotherapeutic agents, which limits the effectiveness of anticancer drugs, is a major problem in cancer therapy. In this study, we used a histone deacetylases inhibitor (HDACi) to establish drug-resistant HCC cells and further analyzed the molecular mechanisms underlying the development of resistance in HCC cells. Compared with the parental cells, HDACi-resistant cells showed high metastatic and pro-survival abilities. Two-dimensional electrophoresis data showed that the cofilin-1 (CFL-1) protein was altered in HDACi-resistant cells and was highly expressed in resistant cells compared with parental cells. The molecular function of CFL-1 is actin depolymerization, and it is involved in tumor metastasis. In this study, we showed that CFL-1 inhibition decreased cell migration and increased cell apoptosis in HDACi-resistant cells. We observed that HDACi induced ROS accumulation in cells and apoptosis via promotion of the CFL-1 interaction with Bax and CFL-1 translocation to the mitochondria, resulting in cytochrome C release. Importantly, phosphorylation of CFL-1 by activated extracellular signal-regulated kinases 1 and 2 (ERK1/2) confers strong protection against HDAC inhibitor-induced cell injury. p-CFL-1 shows a loss of affinity with Bax and will not translocate to mitochondria, stably remaining in the cytoplasm. These results indicate that phosphorylation to inactivate CFL-1 decreased the chemosensitivity to HDAC inhibitors and resulting in drug resistance of HCC cells.

Melatonin suppresses TPA-induced metastasis by downregulating matrix metalloproteinase-9 expression through JNK/SP-1 signaling in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC), a disease common in the South-East Asian population, has high lymph node metastatic ability. Melatonin, an endogenously produced substance present in animals, plants, fungi, and bacteria, has oncostatic activity via several mechanisms. The molecular mechanisms involved in melatonin-mediated tumor inhibitory potential are not completely defined. Here, we show that melatonin treatment inhibits TPA-induced cell motility by regulating the matrix metalloproteinase-9 (MMP-9) expression in NPC. We also identified the signaling cascade through which melatonin inhibits MMP-9 expression; this involves melatonin regulating the binding activity of the transcription factor specificity protein-1 (SP-1)-DNA. Our mechanistic analysis further reveals that the c-Jun N-terminal kinase/mitogen-activated protein kinase pathway is involved in the melatonin-mediated tumor suppressor activity. Furthermore, the findings indicate a functional link between melatonin-mediated MMP-9 regulation and tumor suppressing ability and provide new insights into the role of melatonin-induced molecular and epigenetic regulation of tumor growth. Thus, we conclude that melatonin suppresses the motility of NPC by regulating TPA-induced MMP-9 gene expression via inhibiting SP-1-DNA binding ability. The results provide a functional link between melatonin-mediated SP-1 regulation and the antimetastatic actions of melatonin on nasopharyngeal carcinoma.

Upregulated microRNA-224 promotes ovarian cancer cell proliferation by targeting KLLN.

Human epithelial ovarian cancer is a complex disease, with low 5-yr survival rate largely due to the terminal stage at diagnosis in most patients. MicroRNAs play critical roles during epithelial ovarian cancer progression in vivo and have also been shown to regulate characteristic of ovarian cancer cell line in vitro. Alterative microRNA-224 (microRNA-224) expression affects human epithelial ovarian cancer cell survival, apoptosis, and metastasis. However, people know little about the effects of microRNA-224 on epithelial ovarian cancer cell proliferation. In the current study, we found that the microRNA-224 expression level of human syngeneic epithelial ovarian cancer cells HO8910 (low metastatic ability) was lower than that of HO8910PM (high metastatic ability). Furthermore, microRNA-224 was confirmed to target KLLN in HO8910 and HO8910PM. The known KLLN downstream target cyclin A was regulated by microRNA-224 in HO8910 and HO8910PM. In addition, overexpression of microRNA-224 enhanced the proliferation abilities of HO8910 and knockdown of microRNA-224 suppressed the proliferation abilities of HO8910PM by KLLN-cyclin A pathway. Our results provide new data about microRNAs and their targets involved in proliferation of epithelial ovarian cancer cells by modulating the downstream signaling.