Yafan Wang

Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis.

Cancer-secreted microRNAs (miRNAs) are emerging mediators of cancer-host crosstalk. Here we show that miR-105, which is characteristically expressed and secreted by metastatic breast cancer cells, is a potent regulator of migration through targeting the tight junction protein ZO-1. In endothelial monolayers, exosome-mediated transfer of cancer-secreted miR-105 efficiently destroys tight junctions and the integrity of these natural barriers against metastasis. Overexpression of miR-105 in nonmetastatic cancer cells induces metastasis and vascular permeability in distant organs, whereas inhibition of miR-105 in highly metastatic tumors alleviates these effects. miR-105 can be detected in the circulation at the premetastatic stage, and its levels in the blood and tumor are associated with ZO-1 expression and metastatic progression in early-stage breast cancer.

FZD7 has a critical role in cell proliferation in triple negative breast cancer

Breast cancer is genetically and clinically heterogeneous. Triple negative breast cancer (TNBC) is a subtype of breast cancer that is usually associated with poor outcome and lack of benefit from targeted therapy. We used microarray analysis to perform a pathway analysis of TNBC compared with non-triple negative breast cancer (non-TNBC). Overexpression of several Wnt pathway genes, such as frizzled homolog 7 (FZD7), low density lipoprotein receptor-related protein 6 and transcription factor 7 (TCF7) was observed in TNBC, and we directed our focus to the Wnt pathway receptor, FZD7. To validate the function of FZD7, FZD7shRNA was used to knock down FZD7 expression. Notably, reduced cell proliferation and suppressed invasiveness and colony formation were observed in TNBC MDA-MB-231 and BT-20 cells. Study of the possible mechanism indicated that these effects occurred through silencing of the canonical Wnt signaling pathway, as evidenced by loss of nuclear accumulation of β-catenin and decreased transcriptional activity of TCF7. In vivo studies revealed that FZD7shRNA significantly suppressed tumor formation, through reduced cell proliferation, in mice bearing xenografts without FZD7 expression. Our findings suggest that FZD7-involved canonical Wnt signaling pathway is essential for tumorigenesis of TNBC, and thus, FZD7 shows promise as a biomarker and a potential therapeutic target for TNBC.

De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer

BackgroundMicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome.MethodsThe pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers.ResultsMore than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients.ConclusionsOur study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.

Pharmacodynamic and pharmacogenomic study of the nanoparticle conjugate of camptothecin CRLX101 for the treatment of cancer

CRLX101 is a nanopharmaceutical consisting of cyclodextrin-based polymer molecule and camptothecin. The CRLX101 nanoparticle is designed to concentrate and slowly release camptothecin in tumors over an extended period of time. Tumor biopsy and blood samples collected from patients with advanced solid malignancies before and after CRLX101 treatment are subjected to immunohistochemistry and pharmacogenomics. The expression of Topoisomerase-1, Ki-67, CaIX, CD31 and VEGF decreased after CRLX101 treatment. The expressions of these proteins are inversely proportional with survival duration of the patients. The Drug Metabolism Enzymes and Transporters (DMET) array shows an allele frequency in patients similar to global populations with none of the SNPs associated with toxicity. The results suggest that the observed lower toxicity is not likely to be due to different genotypes in SNPs. CRLX101 demonstrates a promising anti-tumor activity in heavily pre-treated or treatment-refractory solid tumor malignancies presumably by inhibition of proliferation and angiogenesis correlating with tumor growth inhibition. From the clinical editor: In this cancer treatment study clinical samples collected from patients were subjected to immunohistochemistry and pharmacogenomics. The expressions of key proteins that are inversely proportional with survival duration of the patients decreased after treatment with CRLX101, a camptothecin slow-release nanoparticle conjugate. This anti-tumor activity in heavily pre-treated and treatment resistant solid tumors, promises a novel therapeutic approach.

Berbamine inhibits the growth of liver cancer cells and cancer-initiating cells by targeting Ca2+/calmodulin- dependent protein kinase II

Liver cancer is the third leading cause of cancer deaths worldwide but no effective treatment toward liver cancer is available so far. Therefore, there is an unmet medical need to identify novel therapies to efficiently treat liver cancer and improve the prognosis of this disease. Here, we report that berbamine and one of its derivatives, bbd24, potently suppressed liver cancer cell proliferation and induced cancer cell death by targeting Ca2+/calmodulin-dependent protein kinase II (CAMKII). Furthermore, berbamine inhibited the in vivo tumorigenicity of liver cancer cells in NOD/SCID mice and downregulated the self-renewal abilities of liver cancer–initiating cells. Chemical inhibition or short hairpin RNA–mediated knockdown of CAMKII recapitulated the effects of berbamine, whereas overexpression of CAMKII promoted cancer cell proliferation and increased the resistance of liver cancer cells to berbamine treatments. Western blot analyses of human liver cancer specimens showed that CAMKII was hyperphosphorylated in liver tumors compared with the paired peritumor tissues, which supports a role of CAMKII in promoting human liver cancer progression and the potential clinical use of berbamine for liver cancer therapies. Our data suggest that berbamine and its derivatives are promising agents to suppress liver cancer growth by targeting CAMKII. Mol Cancer Ther; 12(10); 2067–77. ©2013 AACR.

Frequent Overexpression of HMGA2 in Human Atypical Teratoid/Rhabdoid Tumor and Its Correlation with let-7a3/let-7b miRNA

Purpose: Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive pediatric malignancies characterized by biallelic inactivation of the SMARCB1 tumor suppressor gene. We searched for novel genomic aberrations by investigating the copy number and expression alterations of let-7a3/let-7b microRNA (miRNA) and correlated these with expression of high-mobility group AT-hook 2 (HMGA2) oncoprotein, a target of let-7 miRNA family, in 18 AT/RT samples to elucidate potential roles of HMGA2 in the pathogenesis of AT/RT. Experimental Design: Genomic aberrations, let-7a3/let-7b miRNA and HMGA2 expression in AT/RT tissues were identified using quantitative PCR, reverse transcription PCR (RT-PCR), and immunohistochemistry. The impact of let-7b miRNA on HMGA2 expression and the malignant potential of human rhabdoid tumor cell G401 (SMARCB1−/−) were investigated by antisense inhibition and ectopic overexpression studies. Results: The copy number of let-7a3/let-7b miRNA was substantially decreased in 4 of 11 AT/RT samples. A significantly inverse correlation between let-7a3/let-7b miRNA expression and HMGA2 mRNA expression was observed in AT/RT tissues (R = −0.34; P < 0.05). Immunohistochemistry analysis demonstrated that HMGA2 was highly overexpressed in 83.3% (15 of 18) of AT/RT tissues. Restoration of let-7 miRNA or knockdown of HMGA2 expression significantly suppressed proliferation and colony formation, and almost abolished the invasive potential of G401 cells. Conclusion: Reduction of let-7a3/let-7b miRNA may be one of mechanisms leading to overexpression of HMGA2 in AT/RT tissues. HMGA2 oncoprotein plays critical roles in the pathogenesis of AT/RT development; and reconstitution of let-7 miRNA or knockdown of HMGA2 oncoprotein may provide a novel therapeutic strategy for the treatment of patients with AT/RT. Clin Cancer Res; 20(5); 1179–89. ©2014 AACR.

Wnt modulates MCL1 to control cell survival in triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) has higher rates of recurrence and distant metastasis, and poorer outcome as compared to non-TNBC. Aberrant activation of WNT signaling has been detected in TNBC, which might be important for triggering oncogenic conversion of breast epithelial cell. Therefore, we directed our focus on identifying the WNT ligand and its underlying mechanism in TNBC cells.MethodsWe performed large-scale analysis of public microarray data to screen the WNT ligands and the clinical significance of the responsible ligand in TNBC. WNT5B was identified and its overexpression in TNBC was confirmed by immunohistochemistry staining, Western blot and ELISA. ShRNA was used to knockdown WNT5B expression (shWNT5B). Cellular functional alteration with shWNT5B treatment was determined by using wound healing assay, mammosphere assay; while cell cycle and apoptosis were examined by flowcytometry. Mitochondrial morphology was photographed by electron microscope. Biological change of mitochondria was detected by RT-PCR and oxygen consumption assay. Activation of WNT pathway and its downstream targets were evaluated by liciferase assay, immunohistochemistry staining and immunoblot analysis. Statistical methods used in the experiments besides microarray analysis was two-tailed t-test.ResultsWNT5B was elevated both in the tumor and the patients’ serum. Suppression of WNT5B remarkably impaired cell growth, migration and mammosphere formation. Additionally, G0/G1 cell cycle arrest and caspase-independent apoptosis was observed. Study of the possible mechanism indicated that these effects occurred through suppression of mitochondrial biogenesis, as evidenced by reduced mitochondrial DNA (MtDNA) and compromised oxidative phosphorylation (OXPHOS). In Vivo and in vitro data uncovered that WNT5B modulated mitochondrial physiology was mediated by MCL1, which was regulated by WNT/β-catenin responsive gene, Myc. Clinic data analysis revealed that both WNT5B and MCL1 are associated with enhanced metastasis and decreased disease-free survival.ConclusionsAll our findings suggested that WNT5B/MCL1 cascade is critical for TNBC and understanding its regulatory apparatus provided valuable insight into the pathogenesis of the tumor development and the guidance for targeting therapeutics.

Expression of DNA translesion synthesis polymerase η in head and neck squamous cell cancer predicts resistance to gemcitabine and cisplatin-based chemotherapy.

Purpose The development of resistance against anticancer drugs has been a persistent clinical problem for the treatment of locally advanced malignancies in the head and neck mucosal derived squamous cell carcinoma (HNSCC). Recent evidence indicates that the DNA translesion synthesis (TLS) polymerase η (Pol η; hRad30a gene) reduces the effectiveness of gemcitabine/cisplatin. The goal of this study is to examine the relationship between the expression level of Pol η and the observed resistance against these chemotherapeutic agents in HNSCC, which is currently unknown. Methods Sixty-four mucosal derived squamous cell carcinomas of head and neck (HNSCC) from 1989 and 2007 at the City of Hope National Medical Center (Duarte, CA) were retrospectively analyzed. Pretreatment samples were immunostained with anti-Pol η antibody and the correlation between the expression level of Pol η and clinical outcomes were evaluated. Forty-nine cases treated with platinum (n=40) or gemcitabine (n=9) based chemotherapy were further examined for Pol η expression level for comparison with patient response to chemotherapy. Results The expression of Pol η was elevated in 67% of the head and neck tumor samples. Pol η expression level was significantly higher in grade 1 to grade 2 tumors (well to moderately differentiated). The overall benefit rate (complete response+ partial response) in patients treated with platinum and gemcitabine based chemotherapy was 79.5%, where low Pol η level was significantly associated with high complete response rate (p=0.03), although not associated with overall survival. Furthermore, no significant correlation was observed between Pol η expression level with gender, age, tobacco/alcohol history, tumor stage and metastatic status. Conclusions Our data suggest that Pol η expression may be a useful prediction marker for the effectiveness of platinum or gemcitabine based therapy for HNSCC.

Signature miRNAs in colorectal cancers were revealed using a bias reduction small RNA deep sequencing protocol

To explore the role of miRNAs in colorectal cancers (CRC), we have deep sequenced 48 pairs of frozen CRC samples, of which 44 pairs produced high quality sequencing data. By using a combined approach of our bias reduction small RNA (smRNA) deep sequencing protocol and Illumina small RNA TruSeq method for sample bar coding, we have obtained data from samples of relatively large size with bias reduced digital profile results. This novel approach allowed us to validate many previously published results using various techniques to profile miRNAs in CRC tissues or cell lines and to characterize ‘true’ miRNA signatures highly expressed in colon/rectum (CR) or CRC tissues. According to our results, miR-21, a miRNA that is up-regulated in CRC, and miR-143, a miRNA that is down-regulated in CRC, are the two miRNAs that dominated the miRNA population in CR tissues, and probably are also the most important miRNAs in CRCs. These two miRNAs, together with the other eight miRNAs, miR-148a, -194, -192, 200b, -200c, -10b, -26a, and -145, with descending expressing levels, constituted the top 10 highly expressed miRNAs in CR/CRC. Using TaqMan miRNA qPCR, we detected the relative expression of some of the CRC miRNAs in 10 CRC cell lines, validated their dysregulation under cancer condition, and provided possible explanation for their dysregulation, which could be caused by APC, KRAS, or TP53 mutations. We believe these results will provide a new direction in future miRNA-related CRC development studies, and application of miRNAs in CRC diagnosis/prognosis, and therapy.

Abstract 4880: Characterization of genetic concordance between primary tumor cells, circulating tumor cells, and metastatic tumor cells from patients with prostate cancer

Circulating tumor cells (CTCs) which disseminate from the primary tumor site have been found to be a major source of metastatic tumor. However, CTCs are heterogeneous and most CTC DNA is fragmented due to apoptosis. To better understand the molecular pathogenesis of metastasis, it is imperative to obtain single, non-apoptotic CTCs and investigate the genetic concordance between primary tumor cells, CTCs and metastatic tumor cells. Due to the abundance of normal cells present in the blood and in tumor tissue, isolation of single tumor cells or pure cell populations of these cells is extremely difficult. The DEPArray platform is designated to collect single CTCs after Cell-Search enrichment, and to sort pure intact tumor cells from formalin-fixed, paraffin-embedded (FFPE) tissues. Taking advantage of this technology, we have isolated single CTCs from prostate cancer patients. Whole genome amplification was performed and genomes with high integrity were used to evaluate the hotspot mutations in PIK3CA. For comparison, we also made single cell suspensions from FFPE tissue blocks of primary tumors and metastatic tumor in lymph nodes from the same patients. Immunofluorescence staining for cytokeratin (CK) and vimentin (Vim) was performed, followed by isolation of pure tumor cells (CK+), stromal cells (Vim+) and double-positive cells (CK+/Vim+) from both primary tumor and lymph node samples. We plan to do genetic analysis on these cells and compare the results to determine the genetic concordance between primary tumor cells, CTCs and metastatic tumor cells. By using the DEPArray, we have successfully isolated single CTCs, and populations of tumor cells, stromal cells and double-positive cells from primary and metastatic tumors. We will present new data on the genetic concordance between these cells, which will provide new insight into our understanding of metastasis in prostate cancer. Citation Format: Lixin Yang, Linling Chen, Yafan Wang, Jeremy Jones, Yun Yen, Sofia Loera, Raju Pillai, Peiguo Chu, Dennis Weisenburger. Characterization of genetic concordance between primary tumor cells, circulating tumor cells, and metastatic tumor cells from patients with prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4880. doi:10.1158/1538-7445.AM2015-4880