This study compared whole exome sequencing performed on the primary CRC and their corresponding lymph node metastasis to the whole-genome sequence of glioblastoma multiforme downloaded from The Cancer Genome Atlas (TCGA) [16]. We identified 115 variant mutations affecting 110 genes that are unique to primary CRC and putatively involved in lymph node metastasis (Fig. 1c). Focusing on variants with significant biological consequences, we selected the variants annotated as missense, splice site or “in frame deletion”; thus discarding variants annotated as silent or falling in non-coding genome (i.e., 3’ and 5’ UTR, intron...). This resulted in a final set of 31 variants mutations affecting 31 genes with putative role in CRC metastasis (Table 1).
Supported by the literature, these genes can be categorized into the following: genes with an identified role in colorectal cancer metastasis (Fig. 2), genes with an identified role in metastasis in general, genes with a potential role in metastasis, and genes with an unknown role in metastasis.
Seven genes with identified role in colorectal cancer metastasis are ADAM19, FBXW7, AEBP1, CDK8, NAV2, SPTBN1 and ABHD11.
ADAM19 and FBXW7 play a role in epithelial to mesenchymal transition (EMT). ADAM19 gene codes for disintegrin and metalloproteinase domain-containing protein 19. It is found to be overexpressed in advanced stage CRC [17]. When knocked down by siRNA (miR-30c) it can suppress migration and invasion of cells [18]. Its silencing upregulates E-cadherin and inhibits N-cadherin and vimentin, thus suggesting a role in EMT [18]. FBXW7 is a tumor suppressor gene with ubiquitin ligase functions. It tags several oncogenic proteins frequently mutated in CRC and is considered a strong negative prognostic factor [19]. Its mutation causes increase in expression of oncogenes including EMT master regulatory genes such as TWIST-1 and SNAIL-1 that act via the mTOR-signaling pathway [20, 21]. Thus, FBXW7 decreased expression allows migration and invasion of CRC cells and therefore promotes its metastasis [22].
AEBP1 and CDK8 can affect matrix metalloproteinase (MMP). AEBP1 (adipocyte enhancer-binding protein 1) upregulates the expression of matrix metalloproteinase-2, vimentin, and TWIST and downregulates E-cadherin through the nuclear factor-κB pathway thus promotes CRC cell proliferation, migration, and invasion [23]. However, it is found to downregulate genes associated with angiogenesis or endothelial function such as aquaporin 1 (AQP1) and periostin (POSTN) when depleted in human umbilical vein endothelial cells (HUVECs) [24]. CDK8 is a colorectal oncogene that allows cancer cell proliferation by regulating β-catenin activity [25]. In addition, it down regulates MMP by inhibiting TIMP3 through the TGFβ/SMAD pathway [26] and it causes induction of MMP9 in human colon cancer cells via Wnt/b-catenin-driven transcription [26].
Neuron navigator 2 (NAV2) regulates F-actin polymerization through the SSH1L/cofilin-1 pathway, thus it facilitates tumor cell migration and invasion. Hence, its overexpression can promote invasion [27].
βII Spectrin (SPTBN1) is a cytoskeleton protein present in all nucleated cells. Loss of SPTBN1 function has multiple effects that may contribute to CRC metastasis. First, by disrupting the TGF-β pathway through Smad3/Smad4 inactivation [28]. Second by decreasing expression of E-cadherin via increased β-catenin nuclear localization and activation of Wnt-pathway through Kallistatin down regulation [28]. Third, by promoting NFκB nuclear localization by inducing the phosphorylation of TAK1 [28].
ABHD11 is a gene known to regulate miR-1254 through the WNT11 pathway and miR-133a through the SOX4 axis. ABHD11 antisense RNA 1 (ABHD11-AS1) expression is increased in CRC and contributes to a poor prognosis [29, 30].
Five genes with identified role in metastasis are KMT2D, TPH2, KRT34, NR1H2 and OPRL1.
KMT2D is a histone methyltransferase and an epigenetic regulator. It promotes growth and metastasis in prostate cancer by transcriptional activation of LIFR and KLF4 targets [31]. Other studies detected KMT2D mutations in 80% of lymph node metastases of esophageal squamous cell carcinoma [32], and associated with poor survival in 6% of triple negative breast cancer [33]. Currently, it is found to play a role in regulating machinery that governs epithelial mesenchymal plasticity [34].
Tryptophan hydroxylase gene 2 (TPH2) encodes a protein important for serotonin synthesis pathway. The gene is found to be unregulated in brain tissue with breast cancer or Melanoma metastasis [35].
Keratin34 (KRT34) gene plays a role in invasive or early tumorigenesis in invasively growing human skin keratinocyte line-HaSKpwC7 cells [36].
Nuclear receptor subfamily 1, group H, member 2 (NR1H2) (Liver X receptor) gene is associated with poor CRC clinical outcome [37], and advanced stage plays a role in prostate cancer development [38].
Nociceptin receptor (OPRL1) gene that represents the telomeres of the chromosome arms in the P036 assay. Ili et al. found that it is one of the most methylated genes in lymph node metastasis in CRC [37]. Lake et al. showed that it is amplified in metastatic conjunctival melanoma [39].
Two genes with potential role in metastasis are ATG16L1 and SGIP1.
Autophagy related 16 like 1 gene (ATG16L1) an autophagy related gene [40]. Qin et al. showed that ATG16L1 is an immune-related gene, which is associated with poor prognosis in patients with osteosarcoma [41]. Thus defects in this gene might contribute to metastasis.
SGIP1 gene codes for a protein involved in endocytosis in neuronal cells. Disturbance to the physiological process of endocytosis can lead to extra cellular matrix (ECM) degradation that might contribute to metastasis [42]. Elevated levels of SGIP1 methylation showed poorer levels of survival in patients with hepatocellular cancer [43].
Seventeen more genes were detected with unknown role in metastasis. The literature search performed by our team did not identify any data pertaining to their involvement in metastasis.
Metastasis is the culmination of cancer progression; however, brain tumors especially glioblastoma multiforme rarely metastasize outside the central nervous system. Puzzlingly, the central nervous system is devoid of a lymphatic system and the cerebrospinal fluid drains directly into the venous cranial sinuses without passing through any lymphatic channels. Moreover, glioblastoma multiforme malignant cells rarely intravasate into the vascular system even though such tumors are extremely vascularized. Recurrent glioblastoma multiforme usual sites are either in the surrounding surgical cavity or in the contralateral hemispheres. Therefore, the hypothesis is that glioblastoma multiforme rarely metastasize outside the central nervous system due to the absence of a lymphatic drainage system [9]. An argument to the hypothesis is that glioblastoma multiforme causes demise in patients before metastasis due to the increase in intracranial pressure. However, one notes that peripheral nervous system tumors specifically neuroblastoma frequently metastasize to loco-regional lymphatics in support of the notion that peripheral nervous system organs drain through lymphatics.
Though, the main limitation of this study is its small sample size; however, it is to be considered a pilot study for identification of putative mutations involved in CRC lymph node metastasis. We suggest performing similar studies with a larger sample size to confirm the role of these mutations in CRC metastasis to loco-regional lymph nodes.