RESEARCH POSTER PRESENTATION DESIGN © 2012

www.PosterPresentations.com

Alcohol consumption has been

implicated in the pathogenesis of head

and neck squamous cell carcinoma

(HNSCC), though its mechanism is

poorly understood. Long non-coding

RNA (lncRNA) is a noncoding RNA that

has a profound effect on numerous

biological processes including

tumorigenesis and oncogenesis. Our

central hypothesis is that alcohol-

mediated dysregulation of lncRNAs is a

key event in HNSCC pathogenesis.

Utilizing TCGA RNA-seq data from 34

HNSCC patients which included alcohol

drinkers and non-drinkers, we analyzed

the expression levels of lncRNAs to

identify a set of differentially expressed

lncRNAs due to alcohol consumption.

Normal oral keratinocytes were

exposed to ethanol and acetaldehyde

to validate the RNA sequencing results

we obtained. A long-term survival

analysis was then conducted on the

verified lncRNAs utilizing a Cox

Proportional-Hazards regression model

and Kaplan-Meier survival curve. We

identified two lncRNAs: lnc-PSD4-1

and the various splice variants of lnc-

NETO-1 that were differentially

expressed due to alcohol consumption

from RNA-seq analysis of the clinical

data. The oral keratinocytes exposed to

alcohol and acetaldehyde also

demonstrated dysregulation of these

two lncRNAs, validating the RNA-seq

analysis. In addition, low expression of

lnc-PSD4-1:14 showed a strong

correlation with higher survival rates.

lnc-PSD4-1:14 and lnc-NETO-1

potentially play a key role in the early

pathogenesis of HNSCC since they are

dysregulated in both clinical data and

in vitro experiments mimicking the

effects of alcohol use.

Abstract

RNA-seq Analysis

RNA-seq libraries

composed of 34 HNSCC

patients, 17 drinkers and

17 nondrinkers were

obtained from The

Cancer Genome Atlas.

The BEDtools utility

coverageBed was utilized

to generate lncRNA read

counts (integer values of

expression level). These

read counts were then

put through a lncRNA

differential expression

analysis comparing

drinkers versus

nondrinkers.

Materials and MethodsDifferentially Expressed lncRNAs

RNA-Seq differential expression analysis

between drinkers and nondrinkers revealed

11 statistically significant lncRNAs.

In Vitro Verification

Of the 11 lncRNAs identified in the RNA-seq

analysis, two were verified: PSD4-1 (including

PSD4-1:14) and NETO1-1, with both having

increased expression levels in the ethanol and

acetaldehyde treated samples compared to

the non-treated controls.

Long-Term Survival Analysis

Low expressions of PSD4-1:14 were highly

correlated with overall better patient survival

in both univariate and multivariate models.

Results ConclusionsThe increased expression of NETO1-1

and PSD4-1:14 in both HNSCC patient

clinical samples and in vitro models of

alcohol usage suggest that NETO1-1

and PSD4-1:14 may act as promoters

of oncogenes. While further study is

necessary to understand the molecular

mechanisms by which these lncRNAs

work, PSD4-1 and NETO1-1 hold

promise as potential biomarkers, and

therapeutic targets of HSNCC, that

could lead to improvement in the

treatment of this disease.Figure Legend

1) Deng G, Sui G (2013) Noncoding RNA in oncogenesis: a new era of

identifying key players. Int J Mol Sci 14: 18319-18349.

2) Homann N, Jousimies-Somer H, Jokelainen K, Heine R, Salaspuro M

(1997) High acetaldehyde levels in saliva after ethanol consumption:

methodological aspects and pathogenetic implications. Carcinogenesis

18: 1739-1743.

3) Hashibe M, Brennan P, Benhamou S, Castellsague X, Chen C, et al.

(2007) Alcohol drinking in never users of tobacco, cigarette smoking in

never drinkers, and the risk of head and neck cancer: pooled analysis

in the International Head and Neck Cancer Epidemiology Consortium.

J Natl Cancer Inst 99: 777-789.

Acknowledgments

• Calit2 and Qualcomm Institute Summer Scholar Program

• Dr. Jessica Wang-Rodriguez

1Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of California San Diego2 Veterans Administration Medical Center and Department of Pathology, University of California San Diego

Pranav Singh1 Vicky Yu1, Elham Rahimy1, Hao Zheng1, Selena Z. Kuo1, Elizabeth Kim1, Weg M. Ongkeko1.

Jessica Wang-Rodriguez2

RNA-seq analysis identifies key long non-coding RNAs connected to the pathogenesis of alcohol-associated head and neck squamous cell carcinoma

Log(2)FC Log(2)CPM p-value FDR

lnc-SLC39A11-2:7 4.194151 3.878865 1.94E-07 0.001085lnc-SLC39A11-2:5 4.140367 3.885084 2.44E-07 0.001085lnc-SLC39A11-2:6 4.142305 3.884648 2.46E-07 0.001085

lnc-LAMB3-1:1 -2.581367 6.606811 2.54E-06 0.008414lnc-CCL18-1:1 -2.190396 5.522936 5.69E-06 0.015064lnc-NETO1-1:9 3.351077 3.585717 1.56E-05 0.022272lnc-NETO1-1:3 3.364047 3.615927 1.63E-05 0.022272lnc-NETO1-1:2 3.362404 3.616193 1.64E-05 0.022272lnc-NETO1-1:4 3.347929 3.625721 1.68E-05 0.022272lnc-NETO1-1:6 3.34735 3.624247 1.68E-05 0.022272lnc-PSD4-1:14 1.669653 1.020086 2.74E-05 0.032967

lnc-SPANXA2-2:1 2.367287 2.927082 3.36E-05 0.037051lnc-AC002472.13.1-1:1 -1.862271 1.547773 4.26E-05 0.041177

lnc-ERC1-1:2 -3.066857 4.256589 4.98E-05 0.041177lnc-FBXL14-1:1 -3.066857 4.256589 4.98E-05 0.041177lnc-ERC1-1:3 -3.066857 4.256589 4.98E-05 0.041177

lnc-KTN1-AS1-1:6 -2.463648 1.922281 5.81E-05 0.042703

Based upon low expression

Univariate Hazard Ratio (95% CI)

p-value Multivariate Hazard Ratio (95% CI)

p-value

LncRNA PSD4-1:14

0.267150( 0.072234-0.988021)

0.047926 0.236208(0.062212-0.896836)

0.034013

Ethanol/Acetaldehyde Treatments

We treated the normal oral keratinocytes

(OKF4, OKF6) with increasing dosages of 200

proof ethanol: 0%, 0.1%, and 0.3% ethanol by

volume. To represent long-term alcohol use,

we treated the cells every 24 hours with

ethanol diluted in medium for a period of 28

days. We also repeated treatment with

acetaldehyde, the first metabolite of ingested

alcohol in the human body. OKF4 and OKF6

were treated with acetaldehyde with effective

concentrations of 0 µM, 75 µM, 150 µM, 300

µM and 1000 µM in medium for a period of

48 hours with acetaldehyde added every 4

hours and media changed every 8 hours.

Quantitative RT-PCR

In vitro lncRNA expression was calculated by

method of qRT-PCR on the ethanol and

acetaldehyde treated cell lines with GAPDH

acting as the endogenous control for gene

expression.

Survival Data Analysis

Utilizing the original 34 patient cohort and

clinical information provided by the TCGA, we

correlated the expression levels of key

dysregulated lncRNAs with the long-term

survival of the patients. A Cox Proportional-

Hazards regression model was then utilized

to determine both univariate and multivariate

survival hazard ratios for the lncRNAs based

upon a low expression.

References

1) Heatmap depicting normalized lncRNA

expression levels (in the form of counts per

million) across the drinker and non-drinker

cohorts using 34 HNSCC patients. Top 100

differentially expressed lncRNAs shown,

including ones that do not have FDR < .05.

2) Panel of Differentially Expressed lncRNAs

with FDR between drinkers and nondrinkers.

NETO1-1 and lnc-SLC39A11-2 are

represented by different splice variants.

3) qRT-PCR analysis of ethanol-treated and

acetaldehyde cell lines, OKF4 and OKF6,

demonstrate that both PSD4-1 and NETO1-

1 are dysregulated by alcohol. Error bars

represent standard deviation.

4) Survival information for PSD4-1:14 in both

univariate and multivariate models

demonstrates a strong correlation with low

expression of PSD4-1:14 and better overall

survival.

5) Kaplan-Meier survival graph depicting a

correlation between low expression of lnc-

PSD4-1:14 and better survival.

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