Background
For over a century, it has been suggested that abnormal chromosome numbers are a pathogenic event that are associated with and drive cancer development [
1]. Extensive studies have confirmed that virtually all tumor types, including solid (e.g. colorectal cancer [
2]) and liquid (e.g. Hodgkin lymphoma [
3]), exhibit genomic instability that often manifests as chromosome instability (CIN). CIN is a dynamic process that is defined by an increased rate at which whole chromosomes or large parts thereof are gained or lost [
4], consequently aneuploidy is now employed as a metric for CIN. Conceptually, CIN promotes tumor heterogeneity by increasing numerical or structural chromosomal changes that in turn impacts oncogene and/or tumor suppressor gene copy numbers [
5]. Thus, CIN is a driver of the tumorigenic process [
6] and often arises through defects in DNA repair, DNA replication or chromosome segregation [
2,
7‐
9]. Tumors displaying CIN (i.e. an aneuploid karyotype) are associated with poor prognosis, and the rapid acquisition of multidrug resistance [
10‐
12]. Despite the strong correlation between CIN and tumors, very little is known about the underlying aberrant genes and/or mechanisms that account for the CIN phenotype.
A recent body of evidence has begun to emerge suggesting that aberrant sister chromatid cohesion may be a pathogenic event that underlies CIN and drives tumor development. Sister chromatid cohesion is an evolutionarily conserved biological process that ensures the faithful segregation of genomic DNA from mother to daughter cells. Classically, cohesion serves to prevent premature chromosome segregation during mitosis by tethering newly synthesized sister chromatids together prior to entry into anaphase [
13], however it also functions in DNA repair [
14] and gene expression [
15]. Mitotic cohesion is effected by a quaternary complex referred to as cohesin [
13], which is comprised of SMC1A, SMC3, RAD21 and STAG1/STAG2/STAG3. Cohesin is initially loaded onto DNA in G1 by cohesin loaders (NIPBL and MAU2), but isn’t established until S-phase when SMC3 is acetylated by ESCO1 and ESCO2 [
16]. Although the exact mechanism (i.e. structure) by which cohesin tethers the sister chromatids is unclear, it is believed to form a ‘ring-like’ structure that encircles the two DNA strands [
17]. During the initial stages of mitosis (prophase to metaphase), cohesion is first lost along the length of the chromosome arms but is maintained/protected at the centromeres through a Shugoshin-dependent mechanism [
18]. A critical requirement of the metaphase to anaphase transition is the regulated cleavage of the remaining centromeric cohesion prior to chromosome segregation. Cohesion cleavage, specifically RAD21 cleavage, is mediated by Separase (
ESPL1), a protease that is normally held in check by Securin (
PTTG1), which is rapidly degraded via a ubiquitin-mediated process orchestrated by APC/C-CDC20.
DNA re-sequencing efforts have determined that defects in cohesin and cohesion-related genes are implicated in various syndromes and diseases including cancer. Defects in
NIPBL,
SMC1A, and
SMC3 for example, are pathogenic in Cornelia de Lange syndrome, a rare genetic disorder associated with growth and cognitive impairment and a shortened lifespan (reviewed in [
19]). Interestingly, individuals with Cornelia de Lange syndrome do not appear to have a predisposition to develop cancer, but this may be due to the shortened lifespan associated with a severe disease state. Still, rare cases of Wilm’s tumors and liver hemangioendothelioma have been identified in autopsies [
20]. Somatic alterations in cohesion-related genes (e.g.
SMC1A,
SMC3,
STAG2,
RAD21, etc.) have however, been identified in a large number of CIN tumor types. Homozygous deletions, amplifications and single nucleotide polymorphisms, which presumably impact expression and/or function and ultimately sister chromatid cohesion have been identified in various cancers including breast, ovarian, colorectal, lung cancer, melanoma, Ewing’s sarcoma, acute myeloid leukemia, myeloid diseases and endometrial cancers [
2,
7‐
9,
21‐
28]. Indeed, we recently demonstrated that diminished expression of a subset of these genes caused aberrant mitotic cohesion and CIN [
7]. Collectively, these data suggest that altered expression and/or function of key cohesion-related genes are pathogenic events that underlie CIN and thus contribute to the development of cancer. However, it is currently unclear how pervasive and prevalent aberrant cohesion is within tumors, specifically within those that frequently exhibit CIN such as Hodgkin lymphoma (HL).
HL is a malignant proliferation of lymphoid cells that frequently exhibits CIN and extensive cytogenetic analyses have routinely identified numerical (and structural) changes in chromosomes within both HL patient samples and cell lines [
29‐
31]. Although there is evidence implicating aberrant telomere biology in the etiology of HL [
32‐
35], the prevalence of aberrant sister chromatid cohesion in HL has never been examined. In the present study, we examined the prevalence of aberrant sister chromatid cohesion within five common HL cell lines and a lymphocyte control. Using cytogenetic techniques, we first established the chromosome distribution profile and modal chromosome number for each line, and subsequently interrogated mitotic spreads for the presence of aberrant cohesion. Surprisingly, all HL lines exhibited aberrant cohesion and strikingly two lines, L-540 and HDLM-2, harbored cohesion defects in ~50% of all mitotic spreads examined. To identify the underlying mechanism that accounts for the aberrant cohesion, the localization and expression pattern of six key cohesion proteins, SMC1A, SMC3, STAG2, RAD21, Securin and Separase, was evaluated. Although all proteins exhibited the expected nuclear localization patterns within each line examined, there was significant variability in the level of RAD21 expression. Of particular interest, was the observation that RAD21 exhibit the greatest variation within these lines and in fact, it was lowest within L-540 and highest within HDLM-2 cell lines. Our results demonstrate that aberrant cohesion is a common feature of all five HL lines evaluated, and further suggest that aberrant RAD21 expression is a causative agent that contributes to the CIN phenotype, particularly within the L-540 and HDLM-2 cell lines. Collectively, our data support recent findings implicating aberrant cohesion as a significant pathogenic factor in the development of various tumor types, which now includes HL.
Discussion
Evidence has accrued that implicates aberrant sister chromatid cohesion as a pathogenic event that causes CIN, and contributes to the development and progression of cancer [
7]. Despite this information, the prevalence of aberrant cohesion within HL, a tumor type that frequently exhibits CIN, has never been determined. Because of this, we purposefully limited the current study to investigations of aberrant cohesion within HL, and thus, we did not attempt to address the role(s) aberrant cohesion may have on gene expression and/or DNA repair. In the current study, we evaluated a panel of five commonly employed aneuploid HL cell lines and show that each exhibits cohesion defects ranging from 10.2% to 55.4%. Strikingly, two lines, L-540 and HDLM-2, harbor defects in ~50% of all mitotic spreads evaluated. Subsequent semi-quantitative Western blot analyses determined that RAD21 expression varied greatly amongst all HL lines but were highest in HDLM-2 and lowest in L-540.
Collectively, our data demonstrate that cohesion defects are a common feature of all HL lines investigated, but are particularly prevalent within the HDLM-2 and L-540. Our study also suggests that aberrant RAD21 expression may contribute to the high levels of aberrant cohesion in HDLM-2 and L-540 cells. Although our analyses cannot preclude
de novo mutations that impact the functions encoded by the six candidate genes evaluated, data gleaned from the Broad-Novartis Cancer Cell Line Encyclopedia [
44] failed to identify a single somatic mutation in any of the six genes within any of the HL cell lines. Moreover, our expression and localization data show that each protein is expressed and exhibits the expected nuclear-enriched localization pattern indicating that epigenetic silencing and/or mis-localization of the six candidates cannot account for the cohesion defects we observe. It should be noted however, that many additional proteins impact sister chromatid cohesion including cohesion loaders, kinases and those involved in cohesion establishment. For example, we previously identified novel roles for CDC4/FBXW7 (a classical cell cycle regulator) and MRE11A (a DNA repair protein) in cohesion [
7]. Thus, it remains formally possible that aberrant expression of additional cohesion-related proteins may contribute to the aberrant cohesion and CIN observed in the HL cell lines. Indeed, the differences in the prevalence and severity of the PCGs between these lines (see Table
2; predominantly PCG
I in HDLM-2 vs. predominantly PCG
III in L-540) suggest that the underlying aberrant mechanism(s) accounting for the cohesion defects are likely to be distinct. Nevertheless, our findings support a growing body of evidence implicating aberrant cohesion as a contributing and driving factor in the development of various cancers, which based on the current findings, can be expanded to include HL.
It is now becoming apparent that altered expression, function, and/or localization of cohesin and cohesion-related proteins causes aberrant cohesion, CIN and is correlated with various disease states. For example, somatic alterations in genes that regulate sister chromatid cohesion have been identified in a number of tumor types including breast, colorectal lung and ovarian cancers, Ewing’s sarcoma, melanoma, acute myeloid leukemia, and myeloid diseases suggesting that aberrant expression contributes to tumorigenesis [
7,
21‐
24,
26‐
28,
42,
44‐
46]. Four large-scale, gene re-sequencing efforts by the Cancer Genome Atlas (TCGA) Network identified non-synonymous single nucleotide polymorphisms (nsSNPs), homozygous deletions and gene amplifications in breast [
21], colorectal [
22], lung [
24] and ovarian [
23] cancers (Table
3). Most relevant to the current study is the finding that
RAD21 is frequently amplified in two of the four tumor types evaluated.
RAD21 is amplified in 13.9% (67/482 tumors) of breast tumors evaluated [
21], while it is amplified in 18.0% (57/316 tumors) [
23] of ovarian tumors. In breast cancer, correlations have been identified between enhanced RAD21 expression [
47] (and the presence of specific nsSNPs [
48]) and overall breast cancer risks, and RAD21 over-expression is associated with poor prognosis and the acquisition of drug resistance [
47]. Aberrant RAD21 mRNA expression has also been examined in endometrial cancers where Supernat et al. [
49] noted elevated expression strongly correlated with more advanced tumor stages and grades. Finally,
RAD21 is amplified and over-expressed in prostate cancer [
50], and its expression is correlated with invasion and metastasis in oral squamous cell carcinomas [
51]. In agreement with these findings, our data show that RAD21 expression levels are elevated in HDLM-2 and are associated with a high prevalence of aberrant cohesion and CIN. Our results also agree with recent mRNA expression data indicating that RAD21 levels are frequently increased in liquid tumors (Additional file
1: Figure S2). For example, publically available data from the CCLE_expression_Entrez_2012-10-18.res: Gene-centric RMA-normalized mRNA expression data (
http://www.broadinstitute.org/ccle) indicate that RAD21 mRNAs levels from the HL cell lines rank 5
th amongst the 37 different tumor types evaluated (Additional file
1: Figure S2). Collectively, these data strongly suggest that RAD21 over-expression may be a pathogenic event in a variety of tumor types, including HL.
Table 3
Somatic alteration in cohesion-related genes in cancer
A
SMC1A
| 0.8 | 0.0 | 0.0 | 4.2 | 0.0 | 0.0 | 1.1 | 0.0 | 0.0 | 1.3 | 0.0 | 0.0 |
SMC3
| 0.2 | 0.2 | 0.0 | 1.4 | 0.9 | 0.0 | 2.8 | 0.6 | 0.0 | 0.3 | 0.0 | 0.0 |
RAD21
| 0.6 | 0.0 | 13.9 | 2.8 | 0.0 | 1.9 | 1.7 | 0.0 | 0.0 | 0.3 | 0.6 | 18.0 |
STAG2
| 1.2 | 0.0 | 0.0 | 2.8 | 0.0 | 0.0 | 3.4 | 0.0 | 0.0 | 0.9 | 0.0 | 0.0 |
PTTG1
B
| 0.0 | 0.4 | 0.2 | 0.5 | 0.0 | 0.0 | 1.1 | 0.6 | 0.0 | 0.0 | 0.0 | 0.9 |
ESPL1
C
| 1.5 | 0.0 | 0.2 | 3.3 | 0.0 | 0.0 | 4.5 | 0.0 | 0.6 | 0.0 | 0.3 | 0.6 |
It is now becoming clear that RAD21 is an excellent and attractive molecule to study for several reasons. From a tumorigenic perspective altered RAD21 expression is associated with a number of tumor types – over-expression is associated with advanced tumor stage and grade, enhanced invasion and metastasis, and the acquisition of multidrug resistance. However, from a therapeutic targeting perspective, RAD21 is also an attractive candidate. For example, Atienza et al. [
52] recently showed that RAD21 suppression decreases cell growth and enhances cytotoxicity to two chemotherapeutic agents, suggesting that chemically targeting RAD21 may hold therapeutic potential. Consequently, tumors that over-express RAD21, such as HDLM-2, may be sensitive to RAD21 suppression. Thus, of the four members of the cohesin complex members, RAD21 is emerging as a lead candidate that may predict tumor behavior and therapeutic resistance, but it may also represent a candidate therapeutic target. In light of these findings we believe that aberrant RAD21 expression, particularly over-expression in HDLM-2 and perhaps underexpression in L-540 (and L-428), has a role in the development and progression of various tumor types, which we have expanded to include HL. Future studies will be required to elucidate the specific mechanism(s) by which RAD21 and other cohesion genes contribute to the development of various tumor types such as HL.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BVS/KJM conceived the study. BVS/ZL carried out the cytogenetic work. BVS/KJM analyzed and interpreted the results. BVS/KJM wrote the manuscript. All authors read and approved the final manuscript.