Abstract

   We have previously found that sera from Crocodylus porosus contain
   anticancer agents and the treatment of MCF7 cells with this serum
   resulted in the differential expression of 51 genes. The purpose of
   this study was to use in silico analysis to identify genes that might
   be epigenetically modulated in cells treated with crocodile serum and
   to understand the role of potential genes as novel candidates with
   epigenetic therapeutic potential. The findings report five
   proto-oncogenes (TUBA1B, SLC2A1, PGK1, CCND1, and NCAPD2) and two tumor
   suppressor genes (RPLP2, RPL37) as novel therapeutic targets.
   Furthermore, we present a comprehensive overview of relevant studies on
   epigenetic regulation of these genes along with an insight into their
   clinical implications. Therefore, elucidating the molecules present in
   the serum and gut bacteria of reptiles such as crocodiles may offer
   insights into the role of these genes on longevity, health, disease,
   and life expectancy.

   Keywords: epigenetics, DNA methylation, Crocodylus porosus, novel
   therapeutics

1. Introduction

   Crocodiles have shown the ability to adapt, evolve, and survive
   successfully over millions of years, suggesting that we ought to learn
   from these species. Having visited several crocodile farms and
   sanctuaries, we find it intriguing that animals such as crocodiles
   tolerate being routinely exposed to radiation, heavy metals, poor diet,
   pollution, etc., and have survived the catastrophic Cretaceous-Tertiary
   extinction event, which was the sudden mass extinction of
   three-quarters of the plant and animal species on Earth that happened
   approximately 66 million years ago. Yet crocodiles have an extended
   lifespan of up to 100 years and thrive in these environments which
   would be unfavourable to humans [[30]1]. Furthermore, crocodiles have
   also been reported to possess bioactive peptides that revealed
   anti-oxidative, anti-inflammatory, anti-microbial, and anti-cancer
   attributes [[31]1,[32]2]. In this regard, we have recently postulated
   and shown that the sera and gut bacteria of animals such as crocodiles
   (Crocodylus palustris and Crocodylus porosus) and water monitor lizard
   (Varanus salvator), tortoise (Cuora amboinensis kamaroma) as well as
   other species that reside in polluted environments depict anti-tumour
   and antibacterial activities and may contribute to the longevity and
   hardiness of these species [[33]3,[34]4].

   Recently, several studies have shown that epigenetics, or the study of
   heritable phenotypic changes, not dependent on DNA sequences, might
   play a pivotal role in a variety of diseases, for example, neurological
   disorders, cardiovascular diseases, metabolic diseases, and cancer
   [[35]5]. Furthermore, epigenetic drugs are already being utilized for
   some cancers and neurological disorders. Several genetic alterations,
   such as specific gene mutations and chromosomal aberrations that are
   associated with breast carcinogenesis have been well studied. In
   addition to these highly characterized mutations, cancer initiation and
   progression are driven by the combined action of multiple epigenetic
   alterations [[36]6]. The three well-known epigenetic alterations are
   DNA methylation, histone modification, and non-coding microRNA-induced
   modulation of gene expression. Of these, DNA methylation is the least
   complex and the most extensively studied. Hypermethylation of
   nucleotide base cytosine found before guanine, CpG Island (CGI), in the
   promoter regions of a tumor suppressor gene, directly blocks the
   binding of transcription factors essential for gene expression, thus
   rendering loss of expression or gene silencing [[37]6]. However,
   hypomethylation of the promoter region of an oncogene could lead to its
   overexpression and abnormal cell survival [[38]7]. Such alterations
   might be participating in the initiation and progression of breast
   carcinogenesis and can be useful as biomarkers for early detection of
   potential therapeutic targets.

   The purpose of this study was to use in silico analysis to identify
   genes that might be epigenetically modulated in cells treated with
   crocodile serum and to understand the role of potential genes as novel
   candidates with epigenetic therapeutic potential.

2. Methods

2.1. In-Silico Analysis

   All the differentially expressed genes (DEGs) in MCF-7 cells treated
   with crocodile serum were selected from our previous study [[39]8]. The
   MCF-7 cell lines (RRID: CVCL_0031) originated from a female patient
   with breast adenocarcinoma and are ER-positive, PGR-positive,
   HER2-negative (source: Cellosaurus;
   [40]https://web.expasy.org/cellosaurus/CVCL_0031; accessed on 15
   February 2022). Using the criteria described previously and explained
   in the results [[41]9], the initial list of 51 genes was filtered for
   the presence of CGIs in their promoter region. Briefly, the key genes
   were filtered on the basis of the presence of CGI at least 200 bp
   upstream of the transcription start site with GC content of >50%,
   length >200 bp, and ratio >0.6 of observed/expected number of CG
   dinucleotides and on the basis of the number of Gs and Cs in the DNA
   segment. A total of 24 genes were selected, which are suspected to be
   epigenetically regulated. Gene set enrichment analysis was performed
   using several ontological resources to identify the underlying
   pathways, and functional protein-protein interaction was depicted.
   Later, “Genotype-Tissue Expression (GTEx) online portal
   ([42]https://www.gtexportal.org/home/; accessed on 15 February 2022)”
   was used to check the expression of our select genes in normal breast
   tissue. Lastly, for analysis of candidate gene expression and promoter
   methylation in cancer tissue, the “TCGA online portal
   ([43]https://portal.gdc.cancer.gov/; accessed on 15 February 2022)” was
   accessed. Gene expression values were reported as transcripts per
   million (TPM) and methylation levels were presented as beta value. The
   beta values (β) are the estimate of methylation level using the ratio
   of intensities between methylated and unmethylated alleles. β are
   between 0 and 1 with 0 being unmethylated and 1 fully methylated.

2.2. Literature Review

   We present a comprehensive literature review of the genes affected by
   DNA methylation that might lead to breast carcinogenesis, using the
   methods as described previously [[44]10]. Briefly, the Medical Subject
   Headings (MeSH) terms used included “breast cancer” with “epigenetic,”
   “DNA methylation,” in combination with the names of candidate genes. We
   searched SCOPUS, PubMed, Ovid, Web of Science, and Google scholar
   databases. All the relevant studies, that were published in the last
   ten years, were identified and included based on their relevance.

3. Results

3.1. Crocodylus Porosus Serum-Induced Differentially Expressed Targets Genes
in MCF-7 Cells

   We have previously reported a differential expression of 51 genes in
   MCF7 cells treated with Crocodylus porosus serum [[45]8]. Here we
   present the relative mRNA expression of those genes as compared with
   untreated control cells ([46]Figure 1A, [47]Table S1). In these treated
   MCF7 cells 26 genes were downregulated, of which the CCN2 gene was most
   significantly inhibited (p < 0.05). And 25 genes were upregulated, of
   which DDIT4 was most significantly overexpressed (p < 0.05). Pathway
   enrichment analysis of our list of DEGs showed significant enrichment
   of GO pathways related to response to stress and electron transport
   genes ([48]Figure 1B, p < 0.01). This indicated a strong induction of
   stress-related genes and mitochondrial genes-related oxidative
   phosphorylation. Next, we analyzed the GTEx database to investigate the
   expression of these in normal breast tissue. We found that most of the
   mitochondrial genes (such as MT-ND1-4, and MT-CO1-3) that were
   upregulated in serum treated MCF7 cells were already very highly
   expressed in the normal breast tissue. Besides, most of the genes that
   were significantly downregulated in serum treated cells, their
   expression was low in normal breast tissue. However, few of the iron
   regulatory genes (FTH1 and SLC2A1) and ribosomal regulatory genes
   (RPLP2 and RPL37) were expressed in contrast to normal tissues versus
   serum-treated MCF7 cells ([49]Figure 1C). Then we sought to investigate
   the relationship between the proteins coded by these candidate genes,
   and STRING was used for the P-P interaction modeling. Computational
   prediction of direct and indirect functional interaction showed that
   all the candidate proteins, except for four of them, have a significant
   level of interaction with each other. But interestingly, the
   mitochondrial proteins and ribosomal proteins appeared as interacting
   more significantly with similar proteins ([50]Figure 1D). Suggesting a
   strong effect of Crocodylus porosus serum on targeting these molecules.

Figure 1.

   [51]Figure 1
   [52]Open in a new tab

   Crocodylus porosus serum-induced differentially expressed targets genes
   in breast cancer cells. (A). Relative mRNA expression of 51
   differentially expressed genes (DEGs) in breast cancer cells treated
   with crocodile serum versus the control untreated cells. All plotted
   genes were statistically significant in comparison to the control. (B).
   Pathway enrichment analysis using DEGs as input genes. (C). Heatmap
   showing expression of DEGs in normal human breast tissues from GTEx.
   (D). Diagram showing protein-protein interactions amongst the DEGs
   encoded proteins.

3.2. Gene Filtration for Potential Epigenetic Regulation in Breast Cancer

   DNA methylation levels are directly regulating the protein expression
   ([53]Figure 2A). Therefore, firstly we aimed to investigate whether DNA
   methylation-based epigenetic modulation is involved in the regulation
   of these 51 candidate genes. So, we searched the NCBI database for the
   presence of CGI in their promoter regions DNA sequence upstream of the
   transcription start site (GC content of >50%, length >200 bp, and ratio
   >0.6 of observed/expected number of CG dinucleotides). Out of the 51
   genes, 24 genes are having CGI in their promoter region ([54]Figure
   2B,C).

Figure 2.

   [55]Figure 2
   [56]Open in a new tab

   Gene filtration for identification of potential epigenetic regulation
   in breast cancer. (A). Line diagram showing DNA methylation induced
   epigenetic modulation of genes. (B). Flow chart showing the process of
   potential epigenetic target gene filtration. (C). Heatmap showing the
   expression pattern of 24 target genes with CpG islands (CGI) in their
   promoter regions in normal versus TCGA breast adenocarcinoma (BRCA)
   tissue samples.

3.3. Novel Genes Which Might Be the Target of Crocodile Serum

   Further, we analyzed the TCGA breast cancer datasets to check the
   expression of these genes in tumour versus normal tissues. Based on the
   gene expression and promoter methylation levels (upregulation and
   hypomethylation or downregulation and hypermethylation), seven (RPLP2,
   RPL37, TUBA1B, SLC2A1, PGK1, NCAPD2, and CCND1) genes were filtered as
   highly likely to be epigenetically regulated and a potential epigenetic
   target of active anticancer molecules presents in Crocodylus porosus
   serum ([57]Figure 2B). The names and functions of the shortlisted genes
   which might be epigenetically regulated via crocodile serum in breast
   cancer cells are presented in [58]Table 1 and [59]Supplementary Table
   S1. TUBA1B, SLC2A1, PGK1, CCND1, and NCAPD2 have been overexpressed in
   breast cancer tissues and their promoter region was hypomethylated
   ([60]Figure 3A–E; The graph on top shows gene expression and the graph
   at the bottom shows methylation beta value). Suggesting an epigenetic
   mediated tumor-promoting function of these genes.

Table 1.

   Details of the genes highly likely to be epigenetically regulated in
   crocodile serum treated breast cancer cells.
   Gene Symbol Genamee n Function (genecards.org) Reference(s) Related to
   Breast Cancer
   TUBA1B Tubulin Alpha 1b Involved in cell cycle spindle assembly and
   chromosome separation [[61]11,[62]12,[63]13,[64]14,[65]15]
   SLC2A1 Solute Carrier Family 2 Member 1 Glucose transporter responsible
   for constitutive glucose uptake
   [[66]16,[67]17,[68]18,[69]19,[70]20,[71]21,[72]22]
   PGK1 Phosphoglycerate Kinase 1 Participates in energy production via
   glycolysis and tumor cell angiogenesis
   [[73]23,[74]24,[75]25,[76]26,[77]27]
   NCAPD2 Non-SMC Condensin I Complex Subunit D2 Required for conversion
   of interphase chromatin into mitotic-like condense chromosomes
   [[78]28,[79]29]
   CCND1 Cyclin D1 Progression of the cell cycle and to induce the Warburg
   effect in cancer cells [[80]30,[81]31]
   RPLP2 Ribosomal Protein Lateral Stalk Subunit P2 An important role in
   the elongation step of protein synthesis [[82]32]
   RPL37 Ribosomal Protein L37 Involved in rRNA processing in the nucleus
   and cytosol [[83]33]
   [84]Open in a new tab

Figure 3.

   [85]Figure 3
   [86]Open in a new tab

   Relative mRNA expression and promoter methylation levels of the genes
   upregulated in cancer cells but downregulated by crocodile serum. (A).
   TUBA1B, (B). SLC2A1, (C). PGK1, (D). CCND1, (E). NCAPD2. (Note: for all
   the box-plots, p < 0.001).

   In breast cancer tissues, RPLP2 and RPL37 were found to be
   downregulated and their promoter region was hypermethylated ([87]Figure
   4A,B). Both genes were significantly up-regulated in MFC7 cells treated
   with crocodile serum ([88]Figure 1A), and crocodile serum treated MCF7
   cells showed reduced proliferation and decreased survival [[89]8].
   Hence, this suggests that these genes might have an epigenetic mediated
   tumor suppressor function. Based on these findings, we believe that
   these genes and their encoded proteins could be novel candidates for
   targeted therapy for breast cancer patients using novel molecules from
   crocodile serum.

Figure 4.

   [90]Figure 4
   [91]Open in a new tab

   Relative mRNA expression and promoter methylation levels of the genes
   downregulated in cancer cells but upregulated by crocodile serum. (A).
   RPLP2, (B). RPL37. (Note: for all the box-plots, p < 0.001).

4. Discussion

   Epigenetic treatment holds great potential in the treatment of several
   diseases such as metabolic disorders, cancer, and cardiovascular and
   neurological diseases [[92]4]. Furthermore, the ability of the
   epigenome to be flexible, has enabled scientists to investigate its
   reversal through designing epigenetic drugs as an approach to
   ameliorate disease phenotypes. For example, recent research has
   revealed that histone modifications are linked to CpG nucleotide
   methylation in DNA, consequently linking several epigenetic
   modifications and regulations [[93]34]. With increasing research,
   therapeutic drug targets continue to be identified and developed with
   more specificity, and greater regulation of epigenetic changes may be
   possible. Many anticancer drugs induce programmed cell death but
   cellular resistance towards many of those chemotherapeutic agents has
   been associated with mutations/deletions of target genes. Hence it is
   essential to search for novel target genes to avoid the toxicity of
   resistant chemotherapy and the unnecessary burden of adverse effects on
   patients. In the search for novel targets, we analyzed the expression
   profile in breast cancer cells treated with crocodile serum. We have
   utilized an in silico approach integrating several human genome
   datasets, such as GTEx, TCGA, and pathway analysis to investigate the
   role of crocodile serum-induced DEGs in human breast cancer tissue
   samples. Treated samples revealed 51 differentially expressed spots
   common to at least two cell lines including proteins involved in
   cytoskeletal organization and cell death. In this study, for the first
   time, we report five oncogenes (TUBA1B, SLC2A1, PGK1, CCND1, and
   NCAPD2) and two tumor suppressors’ genes (RPLP2, RPL37) as novel
   therapeutic targets in breast cancer.

   Tubulin Alpha 1b protein, encoded by the TUBA1B gene, belongs to the
   group of tubulin proteins that are a major constituent of the cellular
   cytoskeleton. These proteins are dynamically involved in the process of
   cell division and replication [[94]11]. TUBA1B was found upregulated in
   the hepatocellular carcinoma (HCC) and mantle cell lymphoma, also its
   increased expression was associated with poor survival [[95]12,[96]13].
   Very recently, a pseudogene-derived upregulation of the TUBA1B gene was
   also reported in human breast cancer samples [[97]14]. Our TCGA
   analysis confirmed this as significant upregulation of TUBA1B genes in
   breast cancer tissues, suggesting its potential oncogenic role.
   Moreover, a genome-wide study reported DNA hypomethylation-induced
   overexpression of the TUBA1B gene in HCC tissue samples [[98]15].
   Inconsistent, our analysis showed promoter hypomethylation of the
   TUBA1B gene in breast cancer tissues confirming its epigenetic
   regulation. Since microtubule proteins play an important role in the
   regulation of cell division, targeting TUBA1B as anticancer therapy
   could lead to a disruption of microtubules causing cell cycle arrest
   and apoptosis.

   The SLC2A1 gene provides instructions to encode a protein called the
   glucose transporter protein type 1 (GLUT1). The GLUT1 protein is a
   membrane-bound receptor that allows the transport of glucose into cells
   for use as fuel. The expression of GLUT1 is reported to be
   significantly elevated in highly aggressive triple-negative breast
   cancer subtypes [[99]16]. Hence, targeting GLUT1 with novel molecules
   had been a point of investigation by many researchers
   [[100]17,[101]18,[102]19,[103]20,[104]21]. Besides, a recent
   meta-analysis indicated that higher GLUT1 expression is associated with
   poor prognostic and that GLUT1 might be a potential biomarker and
   therapeutic target in breast cancer [[105]22]. We showed that the
   SLC2A1 gene was hypomethylated and upregulated in human breast cancer
   tissues. Moreover, the crocodile serum has a strong inhibitory effect
   on the expression of this gene. Cancer cells require abundant glucose
   to meet the energy demands of rapidly proliferating cells [[106]22],
   hence epigenetically silencing SLC2A1 using crocodile serum provides
   new strategies for the sensitization of cancer cells to
   chemo/radiotherapy.

   The PGK1 gene encodes phosphoglycerate kinase enzyme, which is involved
   in a critical energy-producing process of glycolysis. Recently, PGK1
   expression was reported as a part of a 7-gene signature and part of a
   4-gene signature to predict the survival of breast cancer patients
   [[107]23,[108]24]. Furthermore, a higher expression of PGK1 was
   associated with poor prognosis in breast cancer, as it stimulated
   breast cancer progression and metastases [[109]25,[110]26,[111]27].
   Cancer cells can convert glucose into lactic acid even without a lack
   of oxygen, glucose is also converted to lactic acid by tumor cells, and
   the metabolic characteristics of this aerobic glycolysis is called the
   Warburg effect [[112]22], which has been targeted in the development of
   imaging and therapeutic drugs.

   Non-SMC Condensin I Complex Subunit D2, which is encoded by the NCAPD2
   gene, is a regulatory subunit of the condensin complex. It introduces
   positive supercoils into relaxed DNA and converts nicked DNA into
   positive knotted forms. Which is required for the conversion of
   interphase chromatin into mitotic-like condense chromosomes promoting
   the process of cell division [[113]28]. We could find only one study
   that reported that a higher NCAPD2 protein expression was significantly
   correlated with poor overall survival and worse disease-free survival
   in triple-negative breast cancer. Also, breast cancer cells transfected
   with siRNA for NCAPD2 inhibited its expression and consequently
   inhibited the proliferation and invasion of cancer cells [[114]29]. We
   also showed that TCGA breast cancer tissue samples show significantly
   higher NCAPD2 expression as well as hypomethylation of its promoter
   region while we have already reported a crocodile serum-induced
   significant depletion of NCAPD2, which could significantly suppress the
   proliferation of breast cancer cells probably via failure to complete
   mitosis.

   CCND1 translates into Cyclin D1, which is an important regulator of the
   cell cycle. An in silico study has recently suggested that the
   expression of CCND1 is positively correlated with hormone receptor
   positivity in most types of breast cancers [[115]30]. We also found a
   high expression of CCND1 in human breast cancer tissues compared to
   normal. Another study reported miRNA-mediated epigenetic regulation of
   CCND1 in breast cancer cells [[116]31]. Here, we showed that DNA
   hypomethylation might also be mediating the overexpression of this gene
   and that crocodile serum treatment inducing the silencing of this gene
   could be beneficial.

   RPLP2 and RPL37, both encoding for ribosomal proteins, were identified
   as tumor suppressor genes due to their silencing in breast cancer
   tissues. A proteomic study utilizing bioinformatics analysis on breast
   cancer cell lines identified RPLP2 among other several genes correlated
   with chemotherapy-induced cancer cell death [[117]32]. Similarly, in an
   experimental study on breast cancer patients subjected to neoadjuvant
   chemotherapy RPL37 expression along with a few other genes was shown to
   distinguish responsive from non-responsive tumors [[118]33] suggesting
   protective roles of both genes. Consistent with this, we found that
   crocodile-serum induces upregulation of several ribosomal
   protein-related genes. But the TCGA analysis confirmed hypermethylation
   silencing of only the RPLP2 and RPL37 genes in human breast cancer
   tissues. Making them ideal positive prognostic markers and crocodile
   serum as a novel positive regulator of these anti-tumor genes.

   In conclusion, we report seven genes that might epigenetically be
   regulated in breast cancer tissues and could be a potential target for
   epigenetic therapy. Five of these genes (TUBA1B, SLC2A1, PGK1, CCND1,
   and NCAPD2) are thought to be oncogenes, and the two (RPLP2 and RPL37)
   are believed to be tumor suppressor genes. Also, we believe that the
   anti-cancer effect of crocodile serum might be due to the induction of
   promoter methylation changes, specifically in these genes. Epigenetic
   regulatory pathways comprise an emerging and active area of chemical
   probe discovery and investigational drug development. Also, there are
   many great epigenetic drugs, such as 5′Aza-2-deoxycytidine, an
   inhibitor of DNA methyltransferases in clinical trials for cancer
   therapy. Prompted by such emerging clinical relevance of epigenetic
   drugs, we believe that novel molecules present in the crocodile serum
   could prove to be a potential pleiotropic epigenetic drug, but at the
   same time have a precise gene target. Further studies on the gut
   microbiome of the crocodile [[119]1,[120]3] and the sera and gut
   microbiome of other species that live in polluted environments such as
   the water monitor lizard, may lead to the identification of novel
   epigenetic therapeutic targets, given the abilities of these species to
   be resistant to cancer or infectious diseases, and possess longevity
   [[121]1,[122]3].

5. Conclusions

   For the first time in this study, we used a comprehensive in silico
   approach and identified five proto-oncogenes (TUBA1B, SLC2A1, PGK1,
   CCND1, and NCAPD2) and two tumor suppressor genes (RPLP2, RPL37) as
   novel therapeutic targets against breast cancer.

Acknowledgments