Systems genomics of nucleoporins provides prognostic insights into breast cancer

Full Length Research Article

Systems genomics of nucleoporins provides prognostic insights into breast cancer

Rashid Mehmood1*, Kazuya Jibiki2, Zakeya J. Alsafwani1, Muhammad Naseem3, Noriko Yasuhara2*

Adv. life sci., vol. 9, no. 1, pp. 98-110, May 2022
*- Corresponding Author: Rashid Mehmood (Email: rmehmood@alfaisal.edu)
Noriko Yasuhara (Email: yasuhara@chs.nihon-u.ac.jp)
Authors' Affiliations

 1. Alfaisal University, Riyadh – Saudi Arabia
2. Graduate School of Integrated Basic Sciences, Nihon University, Setagaya-ku, Tokyo, Japan
3. Department of Life and environmental Sciences, College of Health and Natural Sciences, Zayed University, Abu Dhabi, UAE; Department of Bioinformatics, Biocenter, University of Würzburg, Am Hubland, Wuerzburg, Germany
 [Date Received: 29/10/2021; Date Revised: 28/12/2021; Date Published: 25/05/2022]

Abstractaa download_button
Introduction
Methods
Results
Discussion
References 

Abstract

Background: Nucleoporins (Nups) constitute a large group of proteins that are structurally arranged at the nuclear envelope and facilitate the bidirectional movement of molecules across the nuclear membrane. In addition to regulating the shuttling of ribonucleoprotein complexes, RNAs and proteins, various Nups interact with chromatin either directly or indirectly, thus regulating gene expression. Any mutations or expression anomalies of Nups may lead to abnormal localization of critical molecules, or dysregulated expression of genes that they interact with. A comprehensive genomic study encompassing all Nup genes in relation to breast cancer is lacking.

Methods: We used genomic and transcriptomic datasets from Pan-Cancer TCGA (The Cancer Genome Atlas), Genotype-Tissue Expression (GTEx) and microarray platforms and conducted in silico analysis of all the genes encoding nucleoporins that are associated with the Nuclear Pore Complexes (NPCs).  For mutation detection, we used cBioportal; for expression analysis, we used Xena and for patient survival plots, KMPlot was utilized.

Results: The genetic and molecular profile of nucleoporin genes identified multiple mutations and detected aberrant expression in breast cancer. Interestingly, NUP133, AHCTF1, TPR, Nup121L showed simultaneous gene amplification in nearly 10% of breast cancer patients. In addition, deregulated expression of some of the nucleoporins, namely, NUP62, NUP 93, NUP98, NUP155, POM121L12, RAE1, SEC13, TPR were correlated with patient prognosis.

Conclusion: The current study is the first one that unravels a comprehensive molecular and genetic profile of nucleoporins genes in breast cancer and underscores the critical roles of various nucleoporins in cancer progression. The identified molecules may advance our understanding of the etiology of the disease and serve as possible targets for novel therapeutic strategies in cancer.

Keywords: Nuclear Pore Complexes (NPCs); Nucleoporins; Breast cancer; Bioinformatics 

Introduction6th button-01

Nuclear pore complexes (NPCs), embedded in the nuclear envelope, provide a gateway for the movement of molecules in and out of the nucleus. The barrier function of the nuclear envelope is quintessential not only for maintaining the distinct composition of the nucleus but also for the unique functions therein. Two types of movements take place through the NPCs; passive diffusion that involves movements of small sized proteins, metabolites, and ions, and facilitative transport wherein large molecules including ribonucleoprotein complexes, RNAs and proteins cross the nuclear envelope. The facilitative transport requires particular transport receptors, called importins and exportins. Both of these types of transport are mediated by the NPCs [1]. NPCs are megadalton complexes (approximately 60-125MDa) and are composed of ~30 distinct proteins called nucleoporins (Nups), which provide binding sites for transport receptors for the transport of molecules in and out of the nucleus [2].

The NPCs are remarkably complicated structures and are composed of three structurally distinct regions. 1) the cytoplasmic ring from which filaments extend into the cytoplasm, 2) the nuclear basket that extends to the nucleoplasmic faces of the NPCs and, 3) the central framework which makes up the pore [3]. The correct positioning of various Nups in the NPC warrants their structural and functional roles, facilitating the transport of selective molecules [4].  The NPC assembly requires coordination between NUPs. For instance, Nup133, a component of the NPC scaffold, is required for Tpr and Nup153 assembly into the nuclear basket [5].  For the transport of molecules into the nucleus, the karyopherins that bind NLS (Nuclear Localization Signal) containing cargoes interact with FG (Phe-Gly) repeats containing Nups of the NPCs and mediate the translocation of the transport complex [6]. Once into the nucleus, the association between Nups and importins is disengaged by the action of RanGTP by virtue of its very high binding affinity to importins [7]. Conversely, the nuclear export of a number of proteins is mediated by interaction of Exportins with RanGTP, while RanGTP independent export also exist, as in case of mRNAs [8].

In addition to their well-known role in the shuttling of molecules across the nuclear membrane, some of the Nups have been shown to regulate a variety of functions, including RNA processing, transcriptional activation, and chromatin modulation [9-11]. Their widespread functions are strengthened by the fact that some NUPs (i.e. NUP98, NUP62, NUP50, NUP153) are not only localized to the NPCs; instead, they exhibit dynamic features by moving off and on of the NPCs. Their shuttling in and out of the nucleus and interaction with cytosolic and nuclear components point to their unorthodox functions. An interesting correlation was found between the expression levels of genes and their position in the vicinity of the NPCs [12]. The observation that the genomic regions nearby the NPCs are transcriptionally active led to the identification of their roles in transcriptional regulation [13, 14].  NUP53, for instance, was shown to not only bind DNA, but its binding pattern was co-enriched with other transcriptional activation markers, i.e. Histone H4K16 acetylation and RNA polymerase II [15].  Over the years, the roles of a battery of Nups were identified in regulating the chromatin landscape [16].  A subset of the Nups regulate expression of genes implicated in cellular identity thus regulating cellular differentiation and development [17, 18]. Moreover, recently, their roles in viral infection and innate immunity were also described [19, 20]. Therefore, it is not surprising that aberrant expression patterns of various Nups are associated with a wide range of medical conditions [21, 22].

Genomic fusions involving Nups with other partners have been found in hematopoietic and non-hematopoietic malignancies [23, 24].  Additionally, the deregulated expression and mutations of some Nups are also linked with various cancers [25, 26].  Their roles in cancer progression may be due to specifically expediting the nuclear translocation of oncogenes, e.g. E2F1, MYC, thus enhancing their oncogenic activities [27] or through interacting with chromatin, thus regulating chromatin remodeling, and regulating the transcription of genes [28].  In breast cancer, only few studies have looked into the individual Nups for their roles in cellular transformation. NUP88 mRNA overexpression, for instance, has been shown to be associated with high aggressiveness of breast cancer [29].  Inverse correlation of NUP93 expression with the survival of triple-negative breast cancer patients by modulating cytoskeleton remodeling has been recently shown[30].  However, to our knowledge, a comprehensive study of all the nucleoporins cataloging their mutational profile, differential expression in comparison with the normal tissue and their correlation with breast cancer patient survival is lacking.

In the current study, using transcriptomic and genomic datasets from The Cancer Genomic Atlas (TCGA) and microarray platforms, we analyzed all the known genes encoding components of NPCs in breast cancer. Our data suggest that majority of NUPs are mutated/aberrantly expressed in breast cancer, albeit with varying degrees. Genetic anomaly with the highest prevalence was found to be the gene amplification. Additionally, other mutations including deep deletions, base substitutions and multiple alterations were also detected. Importantly, aberrant gene expression of some of the Nups correlated with the patient survival, thus pointing to the implications of NUPS in patients’ prognosis. 

Methods6th button-01

Genetic alterations in Nup genes

In order to identify mutations in various Nups, we utilized TCGA (The Cancer Genome Atlas) data using cBioportal [31 ,32]. At the cBioportal, breast cancer datasets are available from multiple sources. We selected the TCGA Breast Invasive Carcinoma (Firehouse legacy) as it employs over 1000 breast cancer patients. In this category, there are 963 patients that had both mutation and CNA (Copy number alterations) data. Therefore, following genetic alterations were scanned in these 963 breast cancer patients' datasets; 1) Mutations, 2) CNA (copy number alterations). Ethical approval was not needed for the study as all datasets in this study were utilized from public databases (TCGA), and processing of data were according to the TCGA publication guidelines (https://cancergenome.nih.gov/publications/guidelines).

Nups and encoding genes nomenclature

Nucleoporins constitute all the proteins that make up the nuclear pore complexes. However, before their identification as a part of this huge nuclear pore complexes, they adapted different names based on interacting proteins and functions they were associated with. For the sake of consistency, we used Human Genome Organization (HUGO) gene nomenclature committee resource (https://www.genenames.org/). All the genes corresponding to nucleoporins were identified from this HUGO resource and published literature [33]. The list of Nups encoded by the corresponding genes are listed in table 1.

NUPs Interactome and network analysis

We mapped protein-protein interactions among NUPs specific proteins as high confidence interactome retrieved from STRING database (https://string-db.org/) [70]. The list of NUPs genes (tables 1) was given as input identifiers to the mutiproteins search option of the STRING database.  A stringent criterion for the selection of interactions as combined edge score greater than 0.9 (P-values for Bayesian confidence score) resulted in a densely connected network, composed of 35 nodes and 175 edges. For network analysis we used STRING inbuilt analysis function for degree and clustering co-efficient. We only selected experimental interactions and visualized the network in evidence mode as STRING output. The edges denote both functional and physical protein associations among the nodes as opted in the basic settings options.

Gene expression changes compared with the normal tissue

For expression comparison between normal tissue and breast cancer samples, we used Xena [71] which consolidates data from TCGA datasets and GTEX for comparison between the two [72]. The gene list in the table 1 was used to generate heat map. All the genes were incorporated in the UCSC Xena. After launching Xena, we selected the first variable phenotype “main category”. “Gene expression” was chosen in the second Genomic variable, followed by incorporating all the genes encoding Nups. TCGA GTEX datasets was used to generate heatmap. Additionally, box plots for the individual genes were fetched from the “view chart” to make gene expression comparisons between GTEX and TCGA breast cancer patients’ datasets. As Xena utilizes the same pipeline for TCGA and GTEx samples, therefore, both datasets are re-analyzed (using UCSC Toil RNA-seq recompute compendium) to eliminate batch effects [71].

Patient survival plots

Kaplan-Meier Plotter was used to generate Meier-Kaplan plots wherein multiple microarray expression datasets are incorporated from 7462 breast cancer patients [73]. In order to generate overall survival plots, the patients were split by median.  For the best probe selection, “jetset” was selected for the shown time periods. The indicated p value does not include correction for multiple hypothesis testing by default [73].  

Results6th button-01

Mutational landscape of various Nups in breast cancer

In order to delineate the roles of nuclear transport process in breast cancer progression, we recently carried out bioinformatic analysis of TCGA datasets and showed that various genes encoding factors related to nuclear transport are mutated [74]. An equally important aspect of the nuclear transport process is the interaction of transport factors with the components of the NPCs and subsequent translocation to the nucleus. Therefore, mutations in the genes coding for Nups, the proteins that constitute the NPCs, may disrupt/expedite the nuclear transport process. Thus, it is imperative to identify various mutations in this category of genes. Therefore, using TCGA datasets, we characterized mutations in Nup genes and found that majority of the Nup genes were genetically altered in breast cancer patients. The alteration frequency differed markedly (ranging from 0.4% in AAAS to 15% in AHCTF1) (Figure 1) amongst various Nups. Interestingly, gene amplification appeared to be the most prevalent genetic

anomaly. Additional mutations were also detected, including deep deletions, base substitutions and multiple alterations. A synergistic gene amplification in AHCTF1, NUP133, NUP210L and TPR was found in over 10% of patients. Figure 1B shows the frequency of mutations in the components of nuclear envelope based on their location in the nuclear pore. To further expand on the possible consequences of genetic alterations on the nuclear pore architecture and to get functional insights into the NUPs genes, we mapped physical interactions among their encoded proteins. A densely connected network of NUPs proteins was derived from the publicly available STRING database [70]. As shown in the Figure 1C, there are 35 protein nodes in the network that constitute 175 edges with diverse functional implications. Interactions between Nup133, AHCTF1 and Nup210L, that are synergistically amplified, are clearly visible in the interactome. Interestingly, alteration frequency in all Nup genes was found to be in nearly 50% of the patients representing significance of this set of molecules (Supplementary figure 1A). Genetic alterations in Nups genes were detected in all breast cancer subtypes albeit with variable rates and types of genetic alterations. In order to validate our analysis, we used PIK3CA, TP53, CDH1, and GATA3. Consistent with literature, we found high prevalence of mutations in these control genes (Figure 1A).

After establishing that Nup genes are mutated in breast cancer, we stratified breast cancer patients into metaplastic breast cancer, breast invasive ductal carcinoma, breast mixed ductal and lobular carcinoma, breast invasive locular carcinoma, and breast invasive mixed mucinous carcinoma and looked for mutations (Supplementary figure 1B). Additional analysis also identified co-occurrences and mutual exclusivities of these top mutated genes with several Nups (Table 2).  The frequency of genetic alterations was not specific to breast cancer. We utilized TCGA PanCancer Atlas Studies that has datasets from a variety of cancers and identified similar trends in mutations in majority of the cancers. This shows that mutations in Nup genes span a spectrum of cancers (Figure 2).

Nups related genes undergo massive expression anomalies in breast cancer

Recent transcriptomic analysis showed massive transcriptomic deregulation is an important hallmark of cancer. A number of genes are not only aberrantly expressed in breast cancer but may also serve as biomarkers. Therefore, we analyzed RNA levels of all Nup genes compared with the normal tissue. The cancer patient’s data was from TCGA, and for comparison GTEx datasets were used. As can be seen in Figure 3A, several Nup genes showed deregulated expression in breast cancer. In majority of the genes analyzed, overexpression was detected to be major anomaly in cancer patients compared with the samples from GTEx. However, a few genes including AAAS, POM121, and DDX19B showed reduced expression in cancer patients compared with the GTEx cohort. The high expression in TCGA datasets might be associated with high amplification rate, at least partially. Figure 3B shows comparisons of individual gene expression between TCGA and GTEx datasets. Collectively, majority of genes encoding Nups go through massive deregulation in expression in cancer patients.

Nups with correlation with patient overall survival

Having established that genetic aberrations and expression changes in cancer patients prevail significantly, next was to determine if any of these changes in gene expression in Nups have a prognostic potential. Mining various microarray datasets using Meier-Kaplan (KM) Plotter [73], we calculated overall survival (OS) of breast cancer patients based on expression of Nup genes. Out of all the Nup genes tested, NUP62, NUP93, RAE1, NUP155, and SEC13 overexpression showed poor overall survival, while POM121L12, TPR, and NUP98 overexpression were correlated with better patient survival. TOP2A, a well-known marker for poor patient survival is also shown in the figure 4, which, consistent with its established role, showed poor patient OS. All other Nups that did not show any significant association with patient overall survival are shown in Supplementary figure 2. Overall, the analysis identifies several molecules that might have prognostic potential in breast cancer.

Analysis of Mismatches

The analysis of the peak of that sequence was done by using the BioEdit software which revealed the presence of mismatches. The software showed any mismatch in nucleotides, but when we analyzed carefully either the sequence or the peaks of electropherograms, indeed it was miss reading of the genetic analyzer but not a mutation in the exons (fig. 3). Similarly, when we aligned exon-5 sequence with reference sequence (NC_000017). In the alignment at position 12330 revealed a deletion of adenine (Fig 3A). Further through the software analysis, it shows a wide adenine peak (fig 3B). We suspected that this might be due to misreading of the sequence. When we repeated the same sample with reverse primers and shows two adjacent adenines with sharp peaks (fig. 3C) at the same place and there was deletion. Overall, this figure suggests that machine mixed the sequence and read as single nucleotide. In similar context another exon-6 sequence showed a deletion of thymine at position 12500 bp when aligned with reference sequence (fig. 3D). Again, when we checked the electropherogram of the same sequence in Bio edit software. The peak of the thymidine was present, but the software couldn’t read the nucleotide sequence (Fig. 3E), which indicate the error of genetic analyzer. The same sequence was reconfirmed with both forward and reverse primers. Contrary to exon-5 and exon-6 misreading, the other exons show 100% similarity with a reference sequence (fig. 3F & 3G). All these results suggest that there might be no correlation between mutation in exon 5-9 of TP53 and breast cancer in Punjabi ethnic group of Pakistan. However, more studies are required to investigate mutation in TP53 gene in large patient cohort. Apart from this, reporting any mutation we should pay great attention and re-confirm with different tools otherwise fall mutation could be disaster for miss diagnosis of the disease.

 

 

 

 

Figures & Tables


 

Discussion6th button-01

In this study, we describe an extensive genetic and molecular landscape of nucleoporins genes in breast cancer. Our analysis shows that a number of Nup genes are not only mutated but also undergo drastic expression changes in breast cancer. Among the Nup genes, we found that AHCTF1, NUP133, NUP210L and TPR were synergistically altered in around 10% of patients. Nup133 is located in the NPC scaffold forming the Y-complex together with Nup85, SEC13, Nup160, Nup107 [75]. It has been shown that Nup133 is instrumental in formation of the nuclear basket with its role in the Tpr and Nup153 recruitment to the NPC basket [76]. Moreover, Tpr is necessary for the nuclear export of mRNAs and proteins. Thus, if the gene products of NUP133 and TPR genes are synergistically altered, the assembly and the functions of NPC could be significantly altered. AHCTF1, also known as ELYS, is not only necessary for post-mitotic NPC assembly through recruitment of chromatin but also regulates the nuclear size [77]. Therefore, simultaneous changes in the quality and quantity of these NUPs could have a significant impact on the function of the nuclear pores, which may define the behavior of cancer. We recently catalogued genetic and molecular attributes of all the genes encoding nuclear transport factors in breast cancer [74]. A crucial part of the nuclear transport process is the interaction of Nups with the actual transport receptors. Nups constitute a group of proteins that constitute the nuclear pore complexes. Their critical role in regulating movement of molecules across the nuclear membrane is already established [78]. One of the categories of cargo that traverse through the nuclear membrane is the proteins related to regulation of cell cycle. Therefore, the functions of Nups in cell cycle regulation become pivotal. Mislocalization of cell cycle regulators has been linked to various cancers [27]. Our knowledge of individual Nups is rudimentary with limited evidence of NUP43, NUP88 and NUP98 to be drivers of breast cancer [79, 80].  However, an overarching molecular profiling of Nup genes in cancer was lacking. Our study fills this gap by providing comprehensive analysis of all Nup genes in breast cancer.

Most popular and known association of Nups with various leukemiac and  non leukemiac malignancies are through juxtaposition of Nup genes with a wide range of other genes [23, 24]. Nup98, for instance, is frequently identified to be translocated with a number of factors and mediate transcription changes depending on its fusion partner [81]. However, there is little knowledge regarding the role of individual Nups in cancer progression. Their molecular analysis of individual role of Nups is critical as they perform diverse aspects of cell cycle either by regulating the nucleocytoplasmic shuttling of various molecules implicated in cancer or by direct or indirect interaction with chromatin., thus regulating expression of genes with possible implications with cancer. An evidence in support of the first aspect was recently reported in prostate cancer wherein overexpression of POM121 led to enhanced nuclear import of E2F1, MYC, and AR, eventually affecting tumorigenesis, proliferation, and survival in lethal prostate cancer [27]. We identified that, in breast cancer, several other Nups were either mutated or had deregulated expression. Interestingly, some of them are implicated in directing nuclear transport by interacting with transport receptors [82], implicating possible nuclear transport related functions.

In addition to above mentioned functions of Nups, evidence regarding their un-expected role in coordinating cell cycle regulation is being established [83]. A recent report highlighted the role of Nup155 in regulating translation of P21 which, a key downstream effector of P53 response in liver cancer, thus regulating P53 network [84]. Nup155 also emerged as an important modulator of chromatin remodeling by interaction with HDAC4 [85]. More recently, Nup155 has been shown to regulate non-coding RNA network mouse embryonic stem cells (mESCs). Partial loss of Nup155 led to reduction in abundance of miR290–295 cluster with concomitant loss of pluripotency factors in mES cells thus affecting pluripotency [86]. The involvement of a single nucleoporin in a wide variety of signaling networks including pluripotency underscores widespread functions. Our data show that NUP155 is not only significantly upregulated in breast cancer but also correlated with poor patient survival. It remains to be seen if and how individual pathways regulated by Nup155 converge onto the pathogenic phenotype in breast cancer.

Another novel molecule that was identified through our in silico analysis is SEC13. Although SEC13 is generally associated with COPII vesicle for the anterograde movement of molecules from ER to Golgi, its location in the nuclear pore is well established where it is a part of Nup107-160 subcomplex [37]. Additionally, its nucleocytoplasmic shuttling of SEC13 is also reported [87]. In the nucleus, it is also shown to bind kinetochores [40]. Its multi-compartmental distribution indicates a variety of possible functions. Our analysis indicates that it is not only significantly upregulated, but also correlates with poor patient survival, thus warranting deeper exploration.

It is pertinent to note that majority of Nups were upregulated in breast cancer patients (Figure 3).  While expression of most of them is correlated with poor patient survival, thus pointing to their roles as oncogenes, overexpression of some Nups correlated with better over all patient survival. The Nups in this category included Nup98, TPR and POM121L12. The correlation between their overexpression and better patient survival might provide a hint for their possible tumor suppressor functions. Further studies are required to realize their role in cell survival and proliferation by using loss of function and gain of function assays in cellular models.

As nucleoporins feed multiple arms of the cellular physiology, it is challenging to identify what particular altered functions might lead to tumorigenesis. Finding the downstream effects is important as it provides an opportunity to target the specific pathway for therapeutic purpose. e.g. Importazole was used to reduce tumorogenic potential in POM121 overexpressed cells as it was mediated through interaction with importin b [88]. However, individual Nups might have role in the disease progression that is independent of nuclear transport functions, e.g the role of Nup62 in regulating gastric cancer metastasis by regulating Wnt/b-Catenin and TGF-b signaling pathways [89]. In such circumstances, individual Nups and/or the downstream pathways that are altered might have to be unraveled for eventual targeting.

Although inhibitors for nuclear transport receptors are relatively well studied in cancer, specific inhibitors for Nups are equally promising in various malignancies considering their massive deregulation, as exemplified by the current study in breast cancer. The mutations in Nup genes were represented in 50% of the breast cancer patients in addition to deregulated expression representing an important vulnerable point. The repertoire of small molecules perturbing the nuclear pore permeability by disrupting FG interactions is limited to only trans1-2 cyclohexanediol (CHD) and Pitstop2 [90, 91]. Despite their ability to compromise the NPC permeability barrier effectively, their systemic effects on the NPCs may hinder their use in therapeutics. Design and synthesis of small molecules targeting specific components of NPCs remains an arduous task, albeit with potential to pave way for novel therapeutic strategies in breast cancer.

In the present study, we couldn’t find a single mutation in exons 5-9 of the TP53 gene in breast cancer patients of Punjabi ethnicity of Pakistan. The exons 5-9 might have no role in breast cancer progression of the Punjabi ethnic group. There is a need to study a large cohort, including familial cancers and sporadic breast cancer patients.

Authors' Contribution

R.M. and N.Y. conceived and designed the study, interpreted data, and finalized the manuscript;

R.M., M. N., and Z. A. conducted Bioinformatics analysis;

R.M., K.J., M.N., and N.Y. were involved in manuscript writing;

All authors read and approved the manuscript.

Conflict of Interest

The authors declare no competing interests.

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