Annals of Oncology

abstracts
SOX2, NANOG, OCT4, and E2F3 increased. miR-145 also up-regulated the markers of
squamous (p63, TP63, and CK5), glandular (MUC-1, MUC-2, and MUC-5AC), and
neuroendocrine (NSE and UCHL-1) differentiation. Finally, miR-145 expression was
down-regulated in high-grade urothelial carcinomas, but not in low-grade tumors.
Conclusions: The results indicate that miR-145 suppresses syndecan-1 and
consequently up-regulates stem cell factors to induce cell senescence and
differentiation. Our data provide a new mechanism of urothelial carcinogenesis via
miR-145, and show that the related molecules could contribute to the development of
new diagnostic or prognostic markers, as well as potential therapeutic targets.
Legal entity responsible for the study: Nara Medical University School of Medicine,
Nara, Japan
Funding: Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and
Technology, Japan (26462424)
Disclosure: All authors have declared no conflicts of interest.
20P
Analysis of miRNA expression profile induced by zoledronic
acid in breast cancer cells
D. Fanale, V. Amodeo, L. Insalaco, L. Incorvaia, A. Listì, V. Calò, V. Bazan,
A. Russo
Department of Surgical, Oncological and Oral Sciences, Section of Medical
Oncology, University of Palermo, Palermo, Italy
Background: Zoledronic acid (ZOL), a third generation bisphosphonate able to
significantly inhibit bone resorption, is currently used for the treatment of breast
cancer patients with osteolytic bone metastases. Our previous work showed an effective
anticancer role of low-dose ZOL in the inhibition of several processes, such as cell
adhesion, invasion, cytoskeleton remodelling and proliferation, in MCF-7 breast cancer
cell line. The main aim of our study was to investigate the molecular mechanisms and
signaling pathways by which ZOL exerts its anti-tumor effects in breast cancer cells
focusing our attention on miRNA expression profile.
Methods: Using a TaqMan Low Density Array A human microRNA microarray
analysis, the expression profile of 377 miRNAs was analyzed in MCF7 cells treated for
24 h with 10 µM ZOL with respect to untreated cells. In addition, the expression of
miRNAs specifically induced by ZOL was analyzed in MCF-7, MDA-MB-231 and
SkBr3 cells using Real-time PCR analyses.
Results: A subset of miRNAs was shown to be differentially expressed following the
low-dose ZOL treatment, and several cancer-related pathways, including PI3/Akt,
MAPK, Wnt, Jak-STAT, TGF-β, and mTOR signaling, were predicted as potential
targets of these deregulated miRNAs, using the DIANA tool mirPath software. In
particular, a set of 54 miRNAs resulted significantly altered after ZOL exposure, with a
group of them being up- or down-regulated, and others induced or silenced by
treatment. Most of these miRNAs are unexamined in breast cancer cells. These data are
perfectly in agreement with the recent results reported in literature regarding some
miRNAs involved in proliferation, bone metastasis development, invasion and therapy
resistance in breast cancer.
Conclusions: This work establishes, for the first time, a link between anticancer effects
of ZOL and miRNA expression changes, suggesting the involvement of some miRNAs
in molecular pathways mediating ZOL activity in breast cancer.
Legal entity responsible for the study: University of Palermo
Funding: Department of Surgical, Oncological and Oral Sciences of Palermo
Disclosure: All authors have declared no conflicts of interest.
21P
MiR-340 inhibits breast cancer cell progression by revering
EZH2 mediated miRNAs dysregulated expression
Z. Shi, J. Zhang
Breast Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin,
China
Background: The anti-tumor activity of miR-340 has been recently characterized in
breast tumor cells. However, the mechanisms underlying miR-340 induced cell growth
and invasion in triple-negative breast cancer (TNBC) have not been well elucidated.
Methods: In this study, we found that miR-340 expression was negative correlated with
EZH2 expression in TNBC sample tissues and cell lines. Subsequent luciferase reporter
assay confirmed that EZH2 (Enhancer of zeste homolog 2) was a novel molecule target
of miR-340. Upregulated MiR-340 expression levels by mimics transfection
significantly inhibited the MDA-MB-231 and MDA-MB-468 breast cancer cells
proliferation, invasion and migration activity, and also induced more cell apoptosis.
Meanwhile, miR-340 upregulation inhibited the tumor growth in an orthotopic
MDA-MB-231 breast cancer mouse models. Furthermore, we found the reduced EZH2
expression by miR-340 mimics transfection also decreased the DNMT1, H3K27me3,
β-catenin and P-STAT3 expressions, which ultimately resulted in the blockage of
miR-21 activity and the induction of miR-200a/b expressions.
Results: our results identified miR-340 as a tumor suppressor in TNBC, moreover, an
EZH2 medicated regulatory loop was also established. Post-transcriptional suppression
of EZH2 expression not only blocked STAT3 mediated miR-21 trans-activation, but
also reversed the miR200a/b silencing by reducing DNMT1 and H3K27me3
expression.
Conclusions: MiR-21 inhibition and miR-200a/b expression trigged by miR-340
cooperated in the TNBC progression.
Legal entity responsible for the study: N/A
Funding: China National Natural Scientific Fund (81502306),Tianjin Medical
University Research Project (2014KYQ08)
Disclosure: All authors have declared no conflicts of interest.
22P
microRNA-331-3p inhibits cell proliferation and E7 expression
by targeting NRP2 in cervical cancer
T. Fujii, Y. Tatsumi, N. Konishi
Pathology, Nara Medical University, Nara, Japan
Background: Aberrant expression of microRNAs (miRNAs) is involved in the
development and progression of various types of cancers. In this study, we investigated
the role of miR-331-3p in cell proliferation and keratinocyte differentiation in uterine
cervical cancer cells. Moreover, we evaluated whether neuropilin 2 (NRP2) which are
putative target molecules of miR-331-3p regulated the human papillomavirus (HPV)
-related oncoproteins E6 and E7.
Methods: Cell proliferation in the human cervical cancer cell lines SKG-II and HeLa was
assessed using the MTS assay. A functional assay for cell growth was performed using cell
viability and cell cycle analysis. Cellular apoptosis was measured using a TUNEL assay.
Quantitative RT-PCR was used to measure the mRNA expression of the E6, E7, NRP2,
p63, and involucrin (IVL) genes and anti-apoptosis markers bcl2, bclXL, and BAX.
Results: Overexpression of miR-331-3p inhibited cell proliferation, and induced G2/M
phase arrest and apoptosis in SKG-II cells. The luciferase reporter assay of the NRP2
3¢-untranslated region revealed direct regulation of NRP2 by miR-331-3p. Gene
expression analyses using quantitative RT-PCR in both SKG-II and HeLa cells
overexpressing miR-331-3p or suppressing NRP2 revealed down-regulation of E6, E7,
and p63 mRNA and up-regulation of IVL mRNA. We showed that miR-331-3p and
NRP2 were key effectors in cell proliferation by regulating the cell cycle and expression
of E6 and E7, and keratinocyte differentiation by down-regulating p63 and
up-regulating IVL.
Conclusions: Our findings suggest that miR-331-3p has an important role in
regulating cervical cancer cell proliferation, and overexpression of miR-331-3p through
suppression of NRP2 may contribute to keratinocyte differentiation and may have
anti-cancer effects. Our future studies will examine whether miR-331-3p and its target,
NRP2, are useful clinical diagnostic and/or prognostic markers for histological and
cytological examination using tissue specimens and liquid-based cytology in the
screening and diagnosis of cervical cancer.
Legal entity responsible for the study: Nara Medical University School of Medicine,
Nara, Japan
Funding: Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and
Technology, Japan (26462424)
Disclosure: All authors have declared no conflicts of interest.
23P
Radioresistance genes in head and neck cancer
Annals of Oncology
A.O. Naghavi 1 , Y. Kim 2 , J. Caudell 1
1 Radiation Oncology, H. Lee Moffitt Cancer Center University of South Florida,
Tampa, FL, USA, 2 Biostatistics, H. Lee Moffitt Cancer Center University of South
Florida, Tampa, FL, USA
Background: Radiotherapy (RT) is integral in the treatment of head and neck cancer
(HNC). Tumors have varying response to RT. Quantifying gene expression and
correlating it with outcome can identify genes that influence a tumor’s response to
radiation. Our goal is to identify the genes predictive of locoregional recurrence (LRR)
in HNC patients treated with RT.
Methods: Patient data was abstracted from a prospectively maintained institutional
Total Cancer Care (TCC) database and chart review. All microarray chips were
normalized using iterative rank-order normalization method. A supervised Gene Set
Enrichment Analysis (GSEA) was performed to determine whether a priori defined
gene pathways shows statistically significant, concordant differences between groups of
samples obtained before and after radiation therapy. 50 well-annotated hallmark gene
sets were obtained from Molecular signatures database v.5.1 for GSEA analysis. Linear
Models for Microarray Data (LIMMA) was used to identify individual genes with
differential expression between the two groups. To explore survival-associated genes,
Cox proportional hazard regression analysis was performed with time to local-regional
recurrence data of patients who underwent radiotherapy. Further, Spearman’s rank
correlation was calculated between expression and survival fraction after 2Gy RT (SF2)
of cancer cell lines in NCI-60 panel. Two-sided false discovery rate (FDR) adjusted
p-value less than 0.05 is considered statistically significant.
Results: A total of 108 patients were analyzed, of which 49 (45%) were sampled prior
to and 59 (55%) after receiving RT. There were a total of 48 (44%) LRRs. Thirty eight
genes were identified in common with a differential expression in NCI 60, prior receipt
vi6 | abstracts Volume 27 | Supplement 6 | October 2016