A genome-wide screen for microdeletions reveals disruption of polarity complex genes in diverse human cancers - PubMed (original) (raw)

. 2010 Mar 15;70(6):2158-64.

doi: 10.1158/0008-5472.CAN-09-3458. Epub 2010 Mar 9.

Gayatry Mohapatra, Miguel N Rivera, Daniel Winokur, Patricia Greninger, Mai Nitta, Peter M Sadow, Gaya Sooriyakumar, Brian W Brannigan, Matthew J Ulman, Rushika M Perera, Rui Wang, Angela Tam, Xiao-Jun Ma, Mark Erlander, Dennis C Sgroi, James W Rocco, Mark W Lingen, Ezra E W Cohen, David N Louis, Jeffrey Settleman, Daniel A Haber

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A genome-wide screen for microdeletions reveals disruption of polarity complex genes in diverse human cancers

S Michael Rothenberg et al. Cancer Res. 2010.

Abstract

In a genome-wide screen of 684 cancer cell lines, we identified homozygous intragenic microdeletions involving genes encoding components of the apical-basal cell polarity complexes. Among these, PARD3 is disrupted in cell lines and primary tumors from squamous carcinomas and glioblastomas. Reconstituting PARD3 expression in both cell types restores tight junctions and retards contact-dependent proliferation. Searching specifically for small intragenic microdeletions using high-resolution genomic arrays may be complementary to other genomic deletion screens and resequencing efforts in identifying new tumor suppressor genes.

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Figures

Fig. 1

Fig. 1

Deletions in polarity complex genes in human tumor samples. (a) Human tumor cell lines with homozygous deletions in polarity complex genes. (b) Mapping of deleted regions to the domain structure of PARD3. *samples with aberrant transcripts encoding frameshift mutations. **splice site point mutation leads to aberrant splicing.

Fig. 2

Fig. 2

Intragenic PARD3 deletions in human cancer cell lines. (a) Heat map (left) and graphical display (right) demonstrating intragenic, homozygous PARD3 deletions in esophagus and head and neck squamous carcinoma cell lines. Deviations to the left of the red line denoting normal copy number (to ~ log[2] −2) represent deleted probes. (b) PCR of genomic DNA confirms the location of the missing exons. (c) RT-PCR of SCCHN cell line DOK (genomic deletion exons 4–5) reveals a truncated transcript from fusion of exons 3 and 6 (left), leading to a frameshift, stop codon (middle) and loss of PARD3 expression (right).

Fig. 3

Fig. 3

PARD3 mutations in human primary tumors. (a) FISH identifies a heterozygous intragenic deletion within PARD3 in a primary SCCHN (left). Only one of two red signals for BAC RP11-198D23 is present in tumor nuclei (T, upper right panel, 44% nuclei) compared to matched normal tissue bordering the tumor (N, upper left--two signals in 94.6% nuclei). FISH with PARD3 BACs surrounding the deleted region demonstrates two signals for each probe (lower panels). RT-PCR (upper right) detects a truncated transcript, the result of out-of-frame fusion of exons 3 and 9 with loss of the intervening exons (lower right). (b) Homozygous deletion of exons 7–8 and 14–16 in a primary glioblastoma. A and B represent two different primer sets for exons 7 and 15. (c) A somatic G-A mutation present in GBM tumor DNA (asterix) but not DNA from matched normal blood (left sequence traces) leads to a truncated transcript (agarose gel image), the likely result of aberrant splicing that joins sequences within exons 9 and 10, with loss of the intervening sequence (right traces).

Fig. 4

Fig. 4

Effect of gain-of-function of PARD3. (a) Restoration of PARD3 expression in squamous carcinoma and glioblastoma cell lines with endogenous deletions. (b,c) Re-expression of PARD3 relocalizes ZO-1 to cell-cell contacts. Arrows indicate location of PARD3 or ZO-1. Asterixes indicate discontinuous ZO-1 staining at some cell borders in control cells. (d) Restoring PARD3 expression in KYSE-30 SCCE cells retards cell growth. The arrow indicates when the cells first appeared to make cell-cell contacts. Cells were plated at 1e5 cells/well in 6-well plates in triplicate. Every 3–4 days, cells were trypsinized and counted. Mean and standard error of the mean (n=3) for one of two representative experiments is shown. *P=0.004. **P=0.005. ***P=0.018 (alpha = 0.05, two-sided, unpaired student’s t-test).

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