9 research outputs found
Inactivation of SLIT2-ROBO1/2 Pathway in Premalignant Lesions of Uterine Cervix: Clinical and Prognostic Significances
The SLIT2-ROBO1/2 pathways control diverse biological processes, including growth regulation. To understand the role of SLIT2 and ROBO1/2 in cervical carcinogenesis, firstly their RNA expression profiles were screened in 21 primary uterine cervical carcinoma (CACX) samples and two CACX cell lines. Highly reduced expressions of these genes were evident. Concomitant alterations [deletion/methylation] of the genes were then analyzed in 23 cervical intraepithelial neoplasia (CIN) and 110 CACX samples. In CIN, SLIT2 was deleted in 22% samples compared to 9% for ROBO1 and none for ROBO2, whereas comparable methylation was observed for both SLIT2 (30%) and ROBO1 (22%) followed by ROBO2 (9%). In CACX, alteration of the genes were in the following order: Deletion:
ROBO1 (48%) > SLIT2 (35%) > ROBO2 (33%), Methylation:
SLIT2 (34%) > ROBO1 (29%) > ROBO2 (26%). Overall alterations of SLIT2 and/or ROBO1 (44%) and SLIT2 and/or ROBO2 (39%) were high in CIN followed by significant increase in stage I/II tumors, suggesting deregulation of these interactions in premalignant lesions and early invasive tumors. Immunohistochemical analysis of SLIT2 and ROBO1/2 in CACX also showed reduced expression concordant with molecular alterations. Alteration of all these genes predicted poor patient outcome. Multiparous (≥5) women with altered SLIT2 and ROBO1 along with advanced tumor stage (III/IV) and early sexual debut (<19 years) had worst prognosis. Our data suggests the importance of abrogation of SLIT2-ROBO1 and SLIT2-ROBO2 interactions in the initiation and progression of CACX and also for early diagnosis and prognosis of the disease
Alterations of ATM and CADM1 in Chromosomal 11q22.3–23.2 Region are Associated with the Development of Invasive Cervical Carcinoma.
To understand the importance of chr11q22.3–
23.2 region in the development of cervical cancer, we have
studied the genetic and epigenetic alterations of the candidate
genes ATM, PPP2R1B, SDHD and CADM1 in cervical
intraepithelial neoplasia (CIN) and cervical carcinoma
(CACX) samples. Our study revealed low expression and
high alterations (methylation/deletion) (55–59%) of ATM
and CADM1 genes along with poor patient outcome. The
alterations of ATM and CADM1 are associated with the
progression of tumor from CIN to Stage I/II, thus implying
their role in early invasiveness. The two genes, PPP2R1B
and SDHD, lying in between ATM and CADM1, have low
frequency of alterations, and majority of the alterations
are in CACX samples, indicating that their alterations might be associated with disease progression. Expressions (mRNA/
protein) of the genes showed concordance with their
molecular alterations. Significant co-alteration of ATM and
CADM1 points to their synergic action for the development
of CACX. Mutation is, however, a rare phenomenon for
inactivation of ATM. Association between the alteration of
ATM and CHEK1 and poor survival of the patients having
co-alterations of ATM and CHEK1 points to the DNA
damage response pathway disruption in development
of CACX. Thus, our data suggest that inactivation of
ATM–CHEK1-associated DNA damage response pathway
and CADM1-associated signaling network might have an
important role in the development of CACX
RBSP3 is frequently altered in premalignant cervical lesions: clinical and prognostic significance
To understand the importance of frequent deletion of 3p22.3 in cervical carcinogenesis, alterations (deletion/methylation/expression) of the candidate genes STAC, MLH1, ITGA9, and RBSP3, located in the region, were analyzed in 24 cervical intraepithelial neoplasia (CIN) and 137 uterine cervical carcinoma (CACX) samples. In CIN, RBSP3 deletion (48%) and methylation (26%) were high compared with the other genes (4–9%). In CACX, alterations of these genes were as follows: deletion: STAC (54%) > MLH1 (46%) > RBSP3 (45%) > ITGA9 (41%), methylation: RBSP3 (25%) > ITGA9 (24%) > STAC (19%) > MLH1 (13%). Overall, alterations of RBSP3 showed association with CIN, whereas for STAC and MLH1, this frequency increased significantly from CIN → Stage I/II and for ITGA9 from CIN → Stage I/II and also from Stage I/II → Stage III/IV. Quantitative mRNA expression analysis showed differential reduced expression of these genes in CACX concordant to their molecular alterations. The more active RBSP3B splice variant was underexpressed in CACX. RB1 was infrequently deleted in CACX. Concordance was seen between (i) inactivation of RBSP3 and intense p-RB1 nuclear immunostaining and (ii) low/absence of MLH1 expression and its molecular alterations in CACX. In normal cervical epithelium, p-RB1 immunostaining was low in differentiated cells, whereas MLH1 staining was seen in both nucleus and cytoplasm irrespective of differentiation stage. Alterations of the genes were significantly associated with poor prognosis. High parity (≥5)/early sexual debut (#x2264;19 years) coupled with RBSP3 alterations/RB1 deletion predicted worst prognosis. Thus, inactivation of RBSP3 might be one of the early events in cervical carcinogenesis
Clinico-pathological features of cervical lesions.
§<p>According to The international federation of gynecology and obstetrics (FIGO) classification.</p
Immunohistochemical staining patterns of ROBO1, ROBO2 and SLIT2.
<p>(<b>A</b>) In normal cervical epithelium the basal and parabasal layer stained intensely for all the three proteins, whereas the intensity and frequency of stained cells reduced with further differentiation in the spinous layer. (<b>B</b>) In primary CACX expression pattern of these proteins were concordant with respective molecular alterations. (<b>C</b>) In SiHa cells ROBO1 and ROBO2 were membrane localized, whereas SLIT2 was present mostly in the cytoplasm. T: primary CACX sample; scale bars for both 20X and 40X is 50 µm; original magnifications are indicated in parenthesis. Magnification of panel <b>C</b> is 40X.</p
Representative autoradiographs showing deletion and microsatellite size alteration (MA) of cervical lesions, at different marker loci.
<p>(i) LOH: loss of heterozygosity, (ii) MA-1: microsatellite size alteration of one allele. (iii) LOH + MA: loss of one allele and microsatellite size alteration of the other. (iv) Hemizygous (HE) deletion of <i>ROBO2</i> locus as shown by D3S2515. (v) & (vi) HE deletion as shown by exonic markers (EM) from <i>ROBO1</i> and <i>ROBO2</i> respectively, <i>SST</i> used as control. The sample numbers and marker loci are indicated above and below the figure respectively. →: allelic loss, “*”: allelic size alteration. Deletion of <i>ROBO1/2</i> and <i>SLIT2</i> analyzed by microsatellite and exonic markers in (<b>B</b>) CIN and (<b>C</b>) CACX. T: Tumor DNA, N: DNA from normal cervix/PBL. (<b>D</b>) Pattern of deletion and methylation of <i>ROBO1/2</i> and <i>SLIT2</i> during disease progression. Asterisk denotes statistical significance (P<0.05). (<b>E</b>) Overall alteration patterns of the individual genes, <i>SLIT2-ROBO1</i> and <i>SLIT2-ROBO2</i> ligand-receptor pairs, during disease progression. Asterisk denotes statistical significance (P<0.05).</p
Kaplan-Meier analysis of survival (up to 5 years) of CACX patients.
<p>Alterations of (<b>A</b>) <i>SLIT2</i> and/or <i>ROBO1</i> and (<b>B</b>) <i>SLIT2</i> and/or <i>ROBO2</i> ligand-receptor pairs were significantly associated with poor patient outcome [OS]. (<b>C</b>) Representation of Cox Multivariate analyses of genetic, clinical and etiological parameters in predicting outcome of CACX patients. N: total number of samples and P<0.05 denotes statistical significance.</p
Correlation between deletion/methylation and reduced expression (RNA/protein) of <i>ROBO1/2</i> and <i>SLIT2</i> in CIN/CACX.
<p>Samples C1, C2 and C3 are CIN lesions.</p><p>D+/−, Deletion (HE, HM, LOH) positive/negative; M+/−, methylation positive/negative; Del/Meth: Deletion or methylation. ↓: Reduced mRNA expression (≥2 folds); ↑: Increased mRNA expression (≥2 folds); -: Reduced/Increased mRNA expression (<2 folds). nd, not done due to insufficient/scanty paraffin embedded tumor tissue; ND, Fresh tissues unavailable for RNA isolation. *, statistically significant (<i>P</i><0.05).</p
Deletions in Chromosome 4 Differentially Associated With the Development of Cervical Cancer: Evidence of Slit2 as a Candidate Tumor Suppressor Gene
The aim of this study was to locate the candidate
tumor suppressor genes (TSGs) loci in the chromosomal
4p15-16, 4q22-23 and 4q34-35 regions associated
with the development of uterine cervical carcinoma
(CA-CX). Deletion mapping of the regions by microsatellite
markers identified six discrete areas with high frequency
of deletions, viz. 4p16.2 (D1: 40%), 4p15.31 (D2:
35–38%), 4p15.2 (D3: 37–40%), 4q22.2 (D4: 34%),
4q34.2-34.3 (D5: 37–59%) and 4q35.1 (D6: 40–50%).
Significant correlation was noted among the deleted
regions D1, D2 and D3. The deletions in D1, D2, D5 and
D6 regions are suggested to be associated with the cervical
intraepithelial neoplasia (CIN), and deletions in the D2,
D3, D5 and D6 regions seems to be associated with progression
of CA-CX. The deletions in the D2 and D6
regions showed significant prognostic implications
(P = 0.001; 0.02). The expression of the candidate TSG
SLIT2 mapped to D2 region gradually reduced from normal cervix uteri fi CIN fi CA-CX. SLIT2 promoter
hypermethylation was seen in 28% CIN samples and significantly
increased with tumor progression (P = 0.04).
Significant correlation was seen between SLIT2 deletion
and its promoter methylation (P = 0.001), indicating that
both these phenomena could occur simultaneously to
inactivate this gene. Immunohistochemical analysis
showed reduced expression of SLIT2 in cervical lesions
and CA-CX cell lines. Although no mutation was detected
in the SLIT2 promoter region (–432 to + 55 bp), CC and
AA haplotypes were seen in –227 and –195 positions,
respectively. Thus, it indicates that inactivation of SLIT2-
ROBO1 signaling pathway may have an important role in
CA-CX development