Although pituitary adenomas, including NFPAs, are commonly considered slow-growing benign brain tumors, a large number of them exhibit a local invasive behavior. Notwithstanding that transcriptome changes associated with NFPA invasiveness have been extensively studied in the NFPAs, that is unpredictable with the aid of current tumor biomarkers [4]. Therefore, current study for the first time utilized the three-way interaction model to provide insights in to upon the biological pathways as well as critical genes associated with invasive nature in the NFPAs.
The validity of fastLA analysis was confirmed by comparing the observed event rate of X3 position (switch) genes in a wide range of significant fastLA p-values and the random one. As presented in Fig. 1, the observed event rate for switch genes is far greater than random. Such a result means that certain genes occupy most X3 positions in the statistically significant triplets.
The biological relevancy of two statistically significant triplets was confirmed using both GSEA and GRN (see Fig. 3 and Fig. 4). Such results suggest that these two triplets may play a central role in PA invasiveness. In the following, we discussed the relationships between involved genes in such triplets separately.
Relationship between involved genes in triplet Fech, {Safb, Cdk9}
In such triplet, Fech is the switch gene that controls the co-expression relationship between gene pair {Safb, Cdk9}. The protein encoded by the Fech gene is ferrochelatase, which is a crucial enzyme that catalyzes the conversion of protoporphyrin IX (PpIX) to heme. A significant down-regulation of Fech expression was found in several malignancies [25–28], resulting in PpIX accumulation in such tumor cells. Indeed, accumulated PpIX in tumor cells leads to photodynamic therapy as effective adjuvant therapy for treating various cancers through visualizing the extent and margins of tumors, including PA [29].
On the other hand, ferrochelatase is involved in endothelial cell growth and choroidal neovascularization [30]. Pusha and coworker [31] found that inhibition of Fech reduces retinal neovascularization and endothelial cell proliferation in the oxygen-induced retinopathy (OIR) mouse model. Furthermore, they suggested griseofulvin as a Fech-inhibiting drug that could be repurposed to treat retinal neovascularization by blocking pathological tuft formation and revascularized areas of vaso-obliteration. Additionally, the inhibitory effect of griseofulvin is reported in skin carcinogenesis [32], thyroid tumors [33], and the development of multiple hepatomas [34].
Although any direct effect of Fech gene expression on PA invasiveness is not reported until now, the positive association between gene expression of Fech gene and epidermal growth factor receptor (EGFR) as a critical gene involving in PA progression has been reported [35].
The results of GSEA (see Fig. 3) show that such triplet is involved in “mRNA processing” biological process. In the following, we discuss about the importance of “mRNA processing” in PA invasiveness.
mRNA processing and PA invasiveness
Intrinsically, cancer evolves through successive genetic alterations that are advantageous to tumor cells. DNA sequence perturbations, as well as epigenomic disruption, are two significant cancer-related alterations [36]. However, besides the genetic changes, abnormalities in the mRNA processing can also trigger cancer formation and motive tumor progression[37]. Indeed, the mRNA processing known as a post-transcriptional mechanism is a crucial biological process during which pre-mRNA undergoes a series of chemical modifications to form the mature mRNA. Subsequently, mature mRNA can be transported to the cytoplasm and translated into the corresponding protein. Such biological processes comprise three critical steps: removing introns by splicing, cleavage the 3′end of mRNA, and polyadenylation [38]. Approximately forty years after recognizing the RNA processing, it is clear that post-transcriptional mechanisms are disrupted in cancer biology [39, 40]. In other words, mRNA processing is frequently altered in the tumors. These alterations lead to the formation of numerous cancer-specific mRNAs translated to misfunction proteins and/or proteins with changed expression levels. Such changed proteins can result in the activation of oncogenes or the inactivation of tumor-suppressor genes [41, 42].
Moreover, abnormality in mRNA processing can be associated with cancer therapeutic resistance. Pre-mRNA processing factor 4 (PRPF4) is known as a novel therapeutic target for breast cancer treatment. The PRPF4 gene was overexpressed in various breast cancer cell lines. The PRPF4 gene was overexpressed in various breast cancer cell lines. Furthermore, Knockdown of the PRPF4 gene reduced migration and breast cancer invasion via suppressing the p38 MAPK phosphorylation pathway [43]. On the other hand, heterogeneous ribonucleoproteins (hnRNPs) that participate in different steps of pre-mRNA processing are involved in human malignancies and metastasis. Many reports also suggested several hnRNAs as promising therapeutic targets in numerous metastatic cancer types [44]. Furthermore, ubiquitin-specific peptidase 39 (USP39) serves critical roles in mRNA processing [45] and additionally, is involved in tumorigenesis of multiple solid malignancies [46, 47], including human renal cell carcinomas (RCC) [48]. XU and colleagues [48] show that silencing of USP39 by siRNA induced cell apoptosis and decreased invasive capacity of RCC cells. Hence, they suggested USP39 as an oncogenic factor that can play a pivotal role in human RCC treatment. Moreover, pre-mRNA processing factor (PRPF) 4B kinase [49], pre-mRNA processing factor 19 (PRP19) [50, 51], and pre-mRNA processing factor 31 (PRP31) [52] are the other oncogenic factors that are involved in mRNA processing pathway. Furthermore, the central role of the factors mentioned above is reported in previous studies in invasiveness and metastatic of numerous malignancies, including prostate cancer, melanoma, hepatocellular carcinoma, and invasive ovarian cancer.
To the best of our knowledge, there is no direct report on the role of the “mRNA processing” pathway in PA so far. Nevertheless, according the above studies, there is considerable evidence to support that such biological process may be associated with the invasiveness of PA.
Relationship between involved genes in triplet Nkx3-1, {Ckap5, Dlg1}
The other significant triplet is Nkx3-1 as the switch gene that controls the co-expression relationship between gene pair {Ckap5, Dlg1}. The switch gene (Nkx3-1) is a homeodomain transcription factor with tumor suppressor function [53]. Homeobox genes comprise a large family of developmental regulators that are essential for cell differentiation and are often aberrantly expressed in cancer [54]. Furthermore, the Nkx3-1 gene is a marker for diagnosing metastatic tumors [55, 56]; besides, loss of Nkx3-1expression occurs in the early tumorigenesis, suggesting such gene plays a role in malignant initiation [55]. Surprisingly, such evidence is consistent with the concept of disease-related-switch genes.
In specific, dysregulation of Nkx3-1 is known as a biomarker for prostate cancer progression [56–58]. Hereupon, anti-NKX3-1 antibodies are used as a method for diagnosing metastatic prostatic adenocarcinomas. Nevertheless, previous studies reported that loss of Nkx3.1 expression correlates with several other malignancies, including breast cancer [56] and salivary duct carcinoma [59].
To the best of our knowledge, no direct link was reported between the Nkx3-1 gene and the PA, although there is an indirect association. The Fgf-2 gene, that plays a central role in the angiogenesis of invasive PA [60–62], is an upstream regulator of NKX genes [63, 64]. Furthermore, the importance of Fgf-2 was reported in human prostate cancer progression [62]. Therefore, it can be inferred that Fgf-2 might control the angiogenesis procedure by regulating the gene expression level of Nkx3-1.
The other aim of the current study was to comprehensively characterize which biological processes may be involved in the invasiveness of PA.
As shown in Fig. 3, above triplet is involved in “spindle organization” biological process. We discussed about such biological process in the PA invasiveness. See below.
Spindle organization and PA invasiveness
Another enriched biological process is “spindle organization”, which assists the arrangement, assembly, and disassembly of spindle components. The spindle, which belongs to cytoskeletal components, is composed of an array of microtubules and associated molecules that forms between opposite poles of a eukaryotic cell during DNA segregation. Accordingly, the spindle plays a pivotal role in separating duplicated chromosomes apart. Hereupon, the correctness of spindle organization and its associated molecules during cell division is crucial for cell fate determination, tissue organization, and cell development. On the other hand, deregulation of cytoskeletal components is associated with several oncogenic phenotypes, including increased migration and invasion of cancer cells [65–67].
Nucleolar and spindle-associated protein 1 (NUSAP1), a microtubule-binding protein, is selectively expressed in proliferating cells. Moreover, it plays a critical role in spindle microtubule organization [68]. The expression levels of NUSAP1 are increased in the G2 to mitosis transition and then immediately decreased after cell division [69]. Previous studies have reported that dysregulation of NUSAP1 is associated with invasion, proliferation, and migration in several malignancies [70–77], including pituitary adenomas [78]. Additionally, Lee and colleagues [78] showed that the NUSAP1 gene upregulated in 95% of patients with pituitary adenomas using the qRT-PCR technique. On the other hand, a pyrrolopyrimidine-based microtubule-depolymerizing agent (PP-13) reduces the metastatic dissemination of invasive cancer cells. PP-13, through binding to the colchicine site of β-tubulin, disturbs microtubules organization; and consequently induces spindle multipolarity, mitotic cell cycle blockade, and apoptosis [79]. Moreover, Gilson and colleagues [80] illustrated that low concentration PP-13 (130 nmol.L-1) treatment significantly decreased the metastatic invasiveness of human cancer cells. Furthermore, they suggested that PP-13 might be a potential alternative to standard chemotherapy in drug-refractory tumors.
The “spindle organization” is defined as a child term for the “cell cycle” process according to the Gene Ontology Databank categories [81]. Several studies confirmed the significant role of the “cell cycle” in PA invasion and migration. See below.
Zhang and colleagues [82] compared differentially expressed microRNAs (DEMs) in the invasive and non-invasive PA. They report that DEMs were significantly associated with the “cell proliferation” and “cell cycle” pathway. On the other hand, Zheng and colleagues [83] showed that MiR-106b is upregulated in the invasive PA patients compare to non-invasive ones, associated with migration and invasion of pituitary adenoma cells. Moreover, they illustrated the inhibition of miR-106b remarkably suppressed proliferation and migration through the arrest of cell cycles. Some other biological molecules that can affect migration and invasion of PA through disturbing the “cell cycle” process include S100 calcium-binding protein A9[84], cyclin B1[85], Lactate dehydrogenase A[86].
Taken together, above evidence confirms the significant role of “spindle organization” in invasiveness and migration of tumor cells.