Background
The chromatin remodelling (CR) complexes dynamically regulate transcription by using the energy from ATP hydrolysis to reposition nucleosomes and modulate accessibility of specific genes to the transcriptional machinery [
1,
2]. Recently, inactivating mutations in the CR complexes have been identified at high frequency in a variety of tumors, highlighting the widespread role of epigenome alterations in tumor suppression or oncogenic activation [
1]. Integrase interactor 1 (INI1, also known as SMARCB1) is a core subunit of the SWI/SNF ATP-dependent CR complex encoded by the corresponding gene at chromosomal position 22q11.2 [
3‐
5]. SMARCB1/INI1 is ubiquitously expressed in normal cells and can be readily identified by immunohistochemistry.
SMARCB1/INI1 germ-line mutations were first described in the malignant rhabdoid tumors (MRT) of infancy and atypical theratoid/rhabdoid tumors of the central nervous system and define a hereditary condition known as “Rhabdoid predisposition syndrome” [
3‐
6]. Deletions at chromosome 22 or loss of
SMARCB1/INI1 expression have also been implicated in the pathogenesis of additional tumor types: renal medullary carcinomas, epithelioid sarcomas, myoepithelial carcinomas and extraskeletal myxoid chondrosarcomas [
7]. Although SMARCB1/INI1 is the most extensively studied subunit of the SWI/SNF complex, very little is known about its role in the pathogenesis of colorectal cancer (CRC) [
8]. Recently, we reported that
SMARCB1/INI1 inactivation or, alternatively, a genomic rearrangement at the chromosome region 22q12 are involved in Rhabdoid Colorectal Tumor (RCT), a rare and highly aggressive neoplasm of the gastrointestinal tract [
9,
10].
SMARCB1/INI1-deficient mice develop rapidly aggressive undifferentiated sarcomas, implying a cancer-related function [
11]. Notably, in the same mouse model, the conditional inactivation of
TP53 leads to a dramatic acceleration of tumor formation and a wider spectrum of cancers than those seen in
TP53 deficient mice alone [
12]. These results suggest a cooperative effect of both genes to prevent oncogenic transformation and a dominant role of
SMARCB1/INI1 to hamper cancer aggressiveness. Despite the evidence in mouse models, the link between
SMARCB1/INI1 alterations and the molecular changes underlying CRC progression remains still poorly understood. In order to shed light on the biological role of
SMARCB1/INI1, in this study we investigated its expression profile and evaluated the relationship between molecular alterations and clinico-histological markers of dedifferentiated and aggressive colorectal carcinomas. We hypothesize that its assessment might be clinically relevant to predict CRC prognosis.
Discussion
The chromatin remodelling complexes mobilize nucleosomes to expose DNA to the transcriptional machinery. Alterations of these complexes are emerging as a critical step in carcinogenesis; in fact, high-frequency mutations in SWI/SNF members have been found in a variety of cancers by whole genome sequencing [
2]. SMARCB1/INI1 is a core subunit of the SWI/SNF complex and a recognized hallmark for the diagnosis of MRT and other mesenchymal cancers [
4,
7]. Negative SMARCB1/INI1 expression is quite rare in epithelial tumors and none of the studies published so far has addressed its role in colorectal cancer [
7,
17]. Only few SWI/SNF components (
BRM, BRG and
ARID1A) have been reported mutated or deregulated in colon cancer; limited functional insights into the mechanisms of oncogenesis promoted by chromatin remodelling complexes are available so far. Even more, the prognostic significance of a large number of SWI/SNF subunits remains unknown [
17‐
21]. Recently, we found that SMARCB1/INI1 expression was either negative or high in rhabdoid colorectal tumors and in a small group of sporadic CRCs [
9,
10].
In the present study, we investigated the SMARCB1/INI1 expression profiles in a larger CRC series and found that the majority (89%) express SMARCB1/INI1 with two distinct patterns of nuclear positivity, low (56%) and high (33%), respectively. The SMARCB1/INI1 nuclear positivity observed in the low expressing group resembled that detected in 60 normal colon tissues. A small group that accounts for 11% of our CRC series displayed a negative SMARCB1/INI1 immunostaining. Notably, negative SMARCB1/INI1 expression was related to poorly differentiated tumors and high frequency of liver metastases disclosing an association between its altered expression and the CRC subgroups more prone to metastatic spreading. SMARCB1/INI1 negative tumors frequently showed loss of CDX2 and high expression of vimentin, two key markers involved in colonic differentiation and mesenchymal phenotype, respectively. Unexpectedly, enhanced vimentin positivity was also found in the group displaying diffuse SMARCB1/INI1 expression.
SMARCB1/INI1 loss-of-function mutations or haploinsufficiency are recurrent in a variety of tumors, especially with rhabdoid features [
4,
7,
17]. The molecular mechanisms underlying SMARCB1/INI1 protein inactivation in CRC were not explored in the present study; however, in agreement with recent data, we ruled out hypermethylation of the
SMARCB1/INI1 promoter region in our CRC cohort (our unpublished data) [
17,
21,
22]. A recent comprehensive genome-wide analysis on 276 CRCs has identified
SMARCB1/INI1 mutations in less than 1% of cases [
21]. These results suggest that epigenetic events might be responsible for
SMARCB1/INI1 inactivation because mutations alone do not fully explain the frequent variations in expression detected in CRCs. Further investigations are needed to answer this question.
The morphological revision of the slides from the 15 SMARCB1/INI1-negative tumors (7%, 1/15) revealed that only one showed a composite rhabdoid histology. The patient had a very short survival time (1 month), confirming the aggressive nature of this subgroup [
8‐
10,
13]. Unlike others RCTs, we found a
KRAS mutation, no
BRAF mutations nor microsatellite instability. These findings reinforce our previous data, implying that
SMARCB1/INI1 plays a crucial role in later stages of colon carcinogenesis [
4,
9,
10].
The most striking finding of our study is the association between loss of
SMARCB1/INI1 expression and a worse clinical outcome, regardless of the tumor stage and MMR status. Unexpectedly, even SMARCB1/INI1-high expression is an adverse prognostic indicator in comparison with SMARCB1/INI1-low expressing tumors. The reasons for this apparent contradiction are not clear: they might be linked to the specific deregulated cross-talks between chromatin remodelling components, acquisition of mesenchymal markers and genomic alterations such as chromosomal instability (CIN). Although still debated, it has been suggested that
SMARCB1/INI1 could have a critical function in determining aneuploidy [
23]. Indeed, a subgroup of CRCs with enhanced SMARCB1/INI1 expression has a consistent proportion of aneuploid cells, even exhibiting MMR deficiency (our unpublished data); these latter tumors, in fact, typically show a near-diploid karyotype [
8]. Whether and how SMARCB1/INI1 dysfunctions are causally implicated in genomic instability remains controversial. We further investigated the SMARCB1/INI1 prognostic significance by exploring its effect in combination with the
TP53 status. Interestingly, the SMARCB1/INI1
-/p53
+ tumor group is closely correlated with very short survival and liver metastases when compared with other SMARCB1/INI1/p53 combinations, demonstrating a cooperative effect of both genes in restraining cancer aggressiveness in CRC advanced stages [
12]. These results evoke the dramatic increase in tumor formation and metastasis obtained by inactivating
TP53 in
SMARCB1/INI1-heterozygous mice. The clinical relevance of deregulated
SMARCB1/INI1 expression is confirmed in two independent CRC datasets of 226 and 146 patients, respectively, providing support to our findings. By interrogating genome-wide expression data, we identified several genes that were coordinately down- or up-regulated and separated in two distinct clusters. Notably, the top genes of the signature (
BCR, COMT, MIF) map to the long arm of chromosome 22 at the cytogenetic band 22q11.23, closely associated with SMARCB1/INI1. The gene expression signature was confirmed also in CRC cell lines displaying molecular features of enhanced vimentin expression, reduced CDX2 and more mesenchymal phenotype. A chromosomal rearrangement (translocation/deletion) at 22q12 has recently been identified in a RCT and correlated with high SMARCB1/INI1 expression [
9,
10]. A further translocation involving TTC28 at 22q12.1 or focal amplification of multiple genes mapped at 22q12.3 has been reported by the Cancer Genome Atlas Network and correlated with tumor aggressiveness [
21]. Based on these evidences, is tempting to speculate that a number of alterations, such as translocations or amplifications, involving a specific region on the long arm of chromosome 22 might be associated with clinical aggressiveness and a more mesenchymal phenotype.
In conclusion, we demonstrate that SMARCB1/INI1 deficiency, alone or in combination with TP53 mutations, influences the CRC aggressive behavior, regardless of the MMR status. Surprisingly, even SMARCB1/INI1 diffuse expression is associated with poor survival, as confirmed in two independent cohorts of patients. We identify several over-expressed or repressed genes located on chromosome 22, close to SMARCB1/INI1 and coordinately deregulated. Our findings suggest that SMARCB1/INI1 and genetic hot spots mapping to the long arm of chromosome 22 play an important role in tumor metastatic spreading. SMARCB1/INI1 might then be a useful clinical prognostic marker to complement the histological examination and grading and to select patients for adjuvant medical treatments. Mechanistic and larger clinical studies are needed to define how chromatin remodelling components and which specific genomic rearrangements influence the CRC metastatic behavior.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Conceived the ideas for this study MP, AR; pathology analysis AR, EM, ADB; MR, DB; Acquisition, analysis and interpretation of data (acquired and managed patients, provided facilities, carried out the immunohistochemistry studies etc.), MP, AR, CZ, LS, CL, LA, PP, LP, AP, FG, AT, AB, RV; Development of methodology (e.g., statistical analysis, biostatistics, computational analysis) MP; CL and MC; Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): MP, EM, LA, PP, LP, AP, FG, AT, AB, RV, CL, AR; Wrote the manuscript MP; AR and VC; All authors read and approved the final manuscript.