In the present study, we clearly demonstrated genomic heterogeneity between paired primary CRC and CRLM by using a large complement of exhaustive genetic analyses with next-generation sequencing. No other study has analyzed such a completely paired sample of primary CRC and synchronous CRLM. Elucidation of the heterogeneity of microenvironment-related factors on the proliferation, invasion, and metastasis of cancer cells will lead to novel diagnostic and therapeutic targets for CRC in the era of genome-guided personalized cancer treatment.
Recent advances in next-generation sequencing technologies have made it possible to analyze large numbers of sequences, leading to international cancer genome analysis projects such as the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC). CRC can be classified into four gene expression-based subtypes with distinguishing features, the consensus molecular subtypes (CMSs): CMS1 (microsatellite instability immune, 14%), CMS2 (canonical, 37%), CMS3 (metabolic, 13%), and CMS4 (mesenchymal, 23%) [20]. This intertumoral heterogeneity has led to the finding that different subtypes of CRC are represented by different genetic makeup, clinical behavior, pathological features, and responses to treatment [21–23].
In addition to the intertumoral heterogeneity mentioned above, intratumoral heterogeneity relates to the genetic heterogeneity between cancer cells within a single tumor. During carcinogenesis, genetic abnormalities accumulate continually, allowing the cells an increased ability to expand and invade. As a result of this continuous process, cancers become genetically heterogeneous, with an indeterminate number of coexisting genomic clones. These clones have different functional characteristics such as the ability to form metastases or respond to chemotherapy.
Cancer cells survive and proliferate in a microenvironment created by the cells themselves, various stromal cells, and the stromal tissue. The stromal cells that form cancer tissues include fibroblasts, vascular and lymphangial endothelial cells, lymphocytes, and macrophages. Both cells interact with cancer cells, imparting their characteristic biological features on the cancer [24, 25]. Distant metastasis of cancer has also been implicated in interactions between cancer cells and stromal cells in the cancer microenvironment [8, 9].
In this study, we determined that many highly expressed genes by DNA microarray analysis were classified as encoding “matricellular proteins”, which interact with the ECM. Periostin is a secreted adhesion-related protein expressed in the periosteum and periodontal ligaments, which acts as a critical regulator of the formation and maintenance of bone and teeth, and also plays an important role in tumorigenesis [26]. Recent studies have shown that periostin is highly expressed in various human cancers and have suggested that periostin promotes tumor growth and metastasis [27–29]. Moreover, periostin is reported to enhance the metastatic growth of colon cancer by both preventing stress-induced apoptosis in cancer cells and augmenting endothelial cell survival to promote angiogenesis. The expression level of periostin is reported to be noticeably higher in metastatic tumors than that in the matched primary colon cancer [30].
Osteopontin is a multifunctional ECM phosphorylated glycoprotein (glycol-phosphoprotein) classified into the Small Integrin-Binding Ligand N-linked Glycoprotein (SIBLING) family, and is reported to play an important role in the tumorigenesis, progression and prognosis of various cancers by regulating cell-matrix interactions and cell signaling through binding with integrins and CD44 receptors [31–36]. A pooled data analysis showed that high osteopontin expression was significantly associated with high tumor grade, invasion, lymph node metastasis, tumor distant metastasis and poor survival in CRC [37].
Thrombospondin-2 (THBS2) is a member of the ECM glycoproteins that mediate ECM assembly, cell-matrix interactions, degradation of matrix metalloproteinases (MMP)-2 and MMP-9, and interact with multiple cell receptors and growth factors. The implication of THBS2 expression in CRC has been controversial. Several studies reported an inverse correlation between THBS2 expression level and malignancy grade [38, 39]. In contrast, resent studies reported that THBS2 expression in CRC was positively correlated with TNM stage and is a strong prognostic indicator [40, 41].
GPNMB gene is reported to be overexpressed in numerous cancers and is often associated with the metastatic phenotype [42–46]. The extracellular domain of GPNMB interacts with integrins to facilitate the recruitment of immune-suppressive and proangiogenic cells to the tumor microenvironment, thereby enhancing tumor migration and invasion [47]. GPNMB expressed in immune cells such as macrophages and dendritic cells [14,29] may impair T-cell activation to down-modulate anti-tumor immune responses [48–50]. However, the role of GPNMB is complex; it appears to have an inhibitory role in some cancers but may promote metastasis in others.
MGP is an ECM protein containing post-translationally modified γ-carboxyglutamate residues due to vitamin K-dependent carboxylation. MGP was initially thought to be involved in the inhibition of calcification of arteries and cartilage. Further investigation demonstrated that MGP had a wider range of activities, which were dependent of the phosphorylation-carboxylation status, protein expression and variants. Recent studies showed that MGP has a role in tumor angiogenesis by increasing vascular endothelial growth factor gene expression [51–53]. Recently, MGP was reported to be upregulated in a variety of tumors, including ovarian, breast, urogenital and skin cancer. However, in colon and lung cancers, an inverse correlation between MGP expression and survival was observed [54].
In the present study, exhaustive genetic analysis using next-generation sequencing and comparison of immunoreactive factors revealed the complexities of gene expression in CRLM. It is especially notable that CRLM has greater genomic heterogeneity associated with the ECM compared to primary CRC. This result suggests our hypotheses. To proliferate in the liver, which differs environmentally from the original colorectal tissue in which they naturally exist, cancer cells must modify their microenvironment to make it more amenable for survival. A suitable microenvironment cannot be regulated by a single factor; instead, complex factors are involved, especially in metastatic sites. The complexity of intratumor heterogeneity, which we revealed in this study, may be an underlying cause of the resistance to treatment for metastatic disease.
Differences in genomic mutational profiling between primary and metastatic sites have been investigated in several studies [55–57]. However, most studies have used metachronous or unpaired patient samples. Moreover, a limited set of biomarkers was employed to show that metastatic sites have more inherited mutations than primary CRC. These studies have potential biases related to both intra- and intertumoral heterogeneity.
It has been reported that the several kinds of systemic chemotherapy could alter the genomic landscape in several cancers [58, 59]. Therefore, adjuvant chemotherapy has the potential to alter the genomic mutational profiles of recurrent cancers, so that they would differ from those of primary tumors [60]. Previous analyses evaluating the heterogeneity of metachronous tumors could be biased by the administration of chemotherapy between the resection of primary CRC and metastatic sites, potentially producing alterations in genomic clones [55, 61]. Our study eliminates this bias since we selected paired samples of synchronous tumors.
This study has two important limitations. First, the study included a relatively small number of patients. We plan to continue our analysis using an increased number of cases in the future. Next, although the gene expression of thrombospondin-2, GPNMB, and MGP in CRLM was more frequent than in primary CRC according to the DNA microarray analysis, the immunohistochemical analysis revealed no differences in expression. We suggest that the discrepancy between the tissue regions analyzed by DNA microarray and those subjected to immunohistochemical analysis led to this result. The relatively low immunoreactivity scores of GPNMB and MGP may be attributed to the timing of degradation and wash-out of these proteins from the tissue [53].
There are three future perspectives from this study. First, we also obtained data of surgically resected tumor specimens and corresponding peripheral blood samples not only for CRLM, but also pulmonary metastasis, peritoneal metastasis, ovarian metastasis and so on, through WES, cancer gene panel sequencing, fusion gene panel sequencing and microarray-based GEP under the framework of “project HOPE”. Thus, we will be able to perform further investigations using these samples. Second, studied of circulating tumor cells (liquid biopsy) may be useful in establishing early CRLM diagnosis. Additionally, it may lead to better predictive biomarkers to identify patients who might benefit from adjuvant chemotherapy. Third, the genes that were overexpressed in this study have the potential to be new therapeutic targets. For example, administration of anti-periostin antibody significantly inhibited the growth of primary tumors as well as metastatic tumors in a murine model of breast cancer [26]. Osteopontin-inhibition is also reported to be a favorable therapeutic approach to metastatic disease [62–65]. An antibody-drug conjugate targeting GPNMB, called glembatumumab vedotin (CDX-011), is currently in clinical studies for various cancers [66].