CC BY
12
Section 2. General Biology
https://doi.org/10.29013/ELBLS-23-1-10-18
Jessica Xiong,
High School: Adlai E. Stevenson High School IL, USA
Wei Wang,
Dr. Instructorj Beijing University
HNSCC: DIFFERENTIAL GENE EXPRESSION IN PRIMARY VERSUS RECURRENT TUMORS
Abstract. Head and Neck Squamous Cell Carcinoma, also known as HNSCC, is the sixth most common cancer worldwide. Between now and 2030, new cases are anticipated to increase by 30%, totalling approximately 1.08 million new cases annually. Generally, all tumors that originate in the mucosal epithelium lining of the oral cavity, pharynx, larynx, and sinonasal tract are considered part of HNSCC. Most HNSCCs in the oral cavity and larynx develop due to abusive alcohol and tobacco consumption, whereas development of HNSCCs in the pharynx seems connected to human papillomavirus (HPV). Due to the consumption of carcinogen products, such as the areca nut, which local people tend to chew, HNSCC is most prevalent in South Asia and Australia. It is also very prevalent in the United States and Europe due to higher infection rates of HPV. Additionally, HNSCC is known for its genetic instability, needing multiple genetic transformations to occur, which is what this study will focus on.
Keywords: HNSCC, HPV.
1. Background HNSCC usually metastasizes to the lungs or nearby
HNSCC is a typically localized cancer. Com- lymph nodes. pared to other cancers, it spreads to distant parts of Progression of invasive HNSCC usually follows the body more slowly. Development of HNSCC is a certain pattern: "epithelial cell hyperplasia, fol-connected to alcohol abuse, tobacco consumption, lowed by dysplasia (mild, moderate and severe), and prior positive HPV infections. Originating in carcinoma in situ and, ultimately, invasive carcino-the oral cavity (which includes lips, buccal mucosa, ma." Since HNSCC is very heterogeneous, cell of hard palate, anterior tongue, floor of mouth and ret- origin usually depends on "anatomical location and romolar trigone), the nasopharynx, the orophar- aetiological agent (carcinogen versus virus)"; how-ynx (which includes palatine tonsils, lingual tonsils, ever, the most common origin is adult stem cells base of tongue, soft palate, uvula and posterior pha- or progenitor cells, which, after oncogenic trans-ryngeal wall), the hypopharynx (which includes formation, turn into cancer stem cells (CSCs) that the bottom part of the throat, extending from the have self-renewal and pluripotency properties (1). hyoid bone to the cricoid cartilage), and the larynx, Although HNSCC CSCs constitute only 1-3% of
cells in primary tumors, there have been a number of molecular biomarkers with prognostic significance. Of these CD44, CD133, and ALDH1 are the most validated. CD44 is a "cell surface receptor for hyaluronic acid and matrix metalloproteinases (MMPs) and is involved in intercellular interactions and cell migration. HNSCC cells with high levels of CD44 are capable of self-renewal, and CD44 levels in HNSCC tumours are associated with metastasis and a poor prognosis. Similarly, increased levels of the membrane-spanning protein CD133 are associated with HNSCC invasiveness and metastasis. ALDH1 is an intracellular enzyme that converts retinol into retinoic acid, plays a part in cellular detoxification and is a marker for both normal stem cells and CSCs. High levels of ALDH1 expression or activity are associated with self-renewal, invasion and metastasis and may have prognostic significance in HNSCC" [2].
To pinpoint the specific cell of origin, it is necessary to look at the development of second primary tumors (SPTs). In HNSCC, SPTs appear at an extremely high rate after the diagnosis of the primary tumor, and they are frequently lethal. The development of SPTs reflects CSCs arising from independent oncogenic transformations by looking at the field cancerization, which "involves the formation of multiple patches of premalignant disease with a higher-than-expected rate of multiple local second primary tumors." This suggests that carcinogens damage large anatomical fields.
Symptoms of HNSCC include persistent sore throat, pain, weakness, or numbness near the head and neck, enlarged lymph nodes, and odd patches or openings in the throat and mouth [3].
Survival rates for HNSCC have improved over the past three decades; the 5-year survival increased from 55% from 1992-1996 to 66% during 20022006. If treated with Surgery, Chemotherapy, Radiation Therapy, Immunotherapy, or Targeted Therapy, survival rates are now 56-62% across all five stages. However, after treatment, 15-50% patients develop
recurrent HNSCC, which is both difficult to treat and a major cause of morbidity. Recurrent HNSCC is difficult to treat because of the loss of effectiveness due to prior treatments and the infiltrative nature of recurrent diseases in the head and neck area. A study done on the most effective treatment for recurrent HNSCC suggests that aggressive treatments, such as surgery and CCRT, reduces deaths of recurrent HNSCC patients most efficiently; however, there is yet a study to be done on the mutated genes responsible for recurrence.
2. Methods
The tools and databases used in this study are publicly available. NCBI's GEO database was used to search for datasets relevant to the objective studied; those with samples separated through arrays were analyzed with GEO2R, while those with samples separated through high throughput sequencing were analyzed with DESeq2. Source Batch Search was used to annotate gene symbols. Results were copied onto Google Sheets, where samples were filtered into upregulated and downregulated according to p-value and fold change. Then, a venn diagram was drawn to find common genes in multiple studies for more accurate results. These genes were then compiled, searched on GenCard Bank, and separated according to their functions.
2.1 Sample Download and Extraction
NCBI's GEO database, a public international
archive storing genomics data submitted by the research community (https://www.ncbi.nlm.nih. gov/geo/), was used. Studies with results relevant to this paper's objective were extracted, and samples were downloaded and separated into two types: array and high throughput sequencing. There were two tumor versus normal samples selected for more accurate results, and one primary versus recurrent sample.
2.2 Array Analysis
GEO2R (https://www.ncbi.nlm.nih.gov/geo/ info/geo2r.html) was used for the array datasets. GEO2R is an interactive web tool available to GEO
datasets with array samples, which allows users to compare two or more of these samples to identify genes that are differentially expressed across set experimental conditions. The results are processed and presented by significance as a table of ordered genes, and graphic plots are available to help visualize differentially expressed genes and assess data set quality.
2.3 High Throughput Sequencing Analysis
DESeq2 (https://bioconductor.org/packages/ release/bioc/html/DESeq2.html) was used for high throughput sequencing analysis. The R program was installed, and the DESeq2 package was downloaded. Then, using code chunks, results were organized into a table.
2.4 Filtering
The resulting tables from both DESeq2 and GEO2R were put separately into google sheets and filtered according to p-value<0.01 and FC<=0.5 (downregulated), FC>=2 (upregulated).
2.5 Commonality Grouping
Resulting gene names were compiled into an online venn diagram tool (https://bioinformatics.psb. ugent.be/webtools/Venn/). The two tumor versus normal upregulated results were inserted, and the common genes were located; this process was repeated for the downregulated genes and the primary versus recurrent genes. The common genes found were separated into upregulated in recurrent tumors and downregulated in recurrent tumors.
2.6 Gene Function Research
GeneCards: The Human Gene Database (https: //www.genecards.org), an online knowledgebase that automatically integrates gene-centric data from ~150 web sources, was used for the research of functions and locations of genes.
3. Results
The basic information ofthe genes were compiled into the table below. In total, there are 9 genes that
regulate physiological processes, 5 genes that regulate tumor-related functions, 5 genes that regulate inflammation, 4 genes that regulate ion-related functions, 3 genes that regulate cell surface adhesion, 2 genes that regulate immune cells, 2 genes that regulate signaling, 2 that regulate antigens, and 5 whose functions are unrelated to any of the others.
1.
Full Name Main Function Protein/gene family Detailed description Up or Down regulated
1 2 3 4 5 6
MET Mesenchymal Epithelial Transition Physiological Processes receptor tyrosine kinase protein family Regulates proliferation, scattering, morphogenesis; reduces lung fibrosis Up
GJA1 Gap Junction Protein Alpha 1 Physiological Processes connexin gene family, encodes protein that's component of gap junctions in the heart Involved in synchronized heart contraction, embryonic development, bladder capacity, and hearing Up
ITGA3 Integrin Subunit Alpha 3 Cell surface adhesion integrin alpha chain protein family n/a Up
HOXD10 Homeobox D10 Physiological Processes Abd-B homeobox protein family Involved in cell differentiation and limb development; part of developmental regulatory system: provides cells with specific positional identities on anterior-posterior axis Up
TCYH3 Tweety Family Member 3 Ion Channels tweety family of proteins Encoded protein is calcium (2+)- activated large conductance chloride (-) channel; responsible for ion channel transport and transport of inorganic cations/anions and amino acids/oligopeptides Up
CA2 Carbonic anhydrase 2 Bone reabsorption isozymes of carbonic anhydrase Essential for bone resorption and osteoclasts differentiation; regulates fluid secretion into anterior chamber of eye; contributes to intracellular pH regulation in duodenal upper villous epithelium during proton-coupled peptide absorption Up
1 2 3 4 5 6
TENM2 Teneurin Transmembrane Protein 2 Physiological Processes, cell surface adhesion, ion channels tenascin Enables cell adhesion molecule and signaling receptor binding activity; involved in calcium-mediated signaling using intracellular calcium source; heterophilic cell-cell adhesion via plasma membrane cell adhesion molecules; retrograde trans-synaptic signaling by trans-synaptic protein complex; involved in neural development by regulating proper connectivity within nervous system Up
CXCL1 C-X-C Motif Chemokine Ligand 1 Inflammation CXC subfamily of chemokines Encoded protein is a secreted growth factor that signals through G-protein coupled receptor and CXC receptor 2; plays role in inflammation and as chemoattractant for neutrophils Up
CDSN Corneodes-mosin Epidermal protein found in cor-neodesmosomes Epidermal barrier integrity Up
LAMB3 Laminin Sub- Physiological basement membrane Mediates attachment, migration, or- Up
unit Beta 3 processes proteins ganization of cells into tissues during embryonic development by interacting e other extracellular matrix components
ITGA6 Integrin Subunit Alpha 6) Cell surface adhesion integrin alpha chain protein family Present in oocytes, involved in sperm-egg fusion; plays structural role in hemidesmosome Up
FMNL2 Formin Like 2 Physiological processes formin-related protein Regulates cell morphology and cy-toskeleton organization; required in cortical actin filament dynamics Up
MMP10 Matrix Metal- Physiological peptidase M10 family Breaks down extracellular matrix in Up
lopeptidase 10 processes of matrix metallopro-teinases (MMPs) normal physiological processes (embryonic development, reproduction, tissue remodeling, and disease processes like arthritis and metastasis)
SLC2A1 Solute Carrier Family 2 Member 1 Glucose transport Solute carrier family Encodes major glucose transporter in mammalian blood-brain barrier; protein mainly found in cell membrane and cell surface, also functions as receptor for HTLV virus I and II Up
B3GALT5 Beta-1,3-Ga-lactosyltrans-ferase 5 Antigens membrane-bound glycoproteins Encoded protein may synthesize typel Lewis antigens, which are elevated in gastrointestinal and pancreatic cancers down
1 2 3 4 5 6
ADH7 Alcohol Dehydrogenase 7 Metabolize substrates class IV alcohol dehydrogenase 7 mu or sigma subunit Most active as retinol dehydrogenase, thus may participate in synthesis of retinoic acid (hormone used for cellular differentiation); catalyzes NAD-dependent oxidation of all-trans-retinol, alcohol, and omega-hydroxy fatty acids down
HPGD 15-Hydroxy-prostaglandin Dehydroge-nase Metabolism of prostaglan-dins, inflammation short-chain non-me-talloenzyme alcohol dehydrogenase protein family Catalyzes NAD-dependent oxidation of hydroxylated polyunsaturated fatty acids; decreases levels ofpro-proliferative prostaglandins such as prostaglandin E2 (whose activity increased in cancer because increase in expression of cyclooxy-genase 2); inactivates resolvins E1, D1, D2, which play roles in inflammation down
SCGB1A1 secretoglobin family 1A member 1 Physiological processes, inflammation secretoglobin family of small secreted proteins Anti-inflammation, inhibition of phospholipase A2, sequestering of hydrophobic ligands down
NUCB2 Nucleobin-din-2 Ions, tumor related calcium binding protein Calcium level homeostasis, eating regulation in hypothalamus, release of tumor necrosis factor from vascular endothelial cells; non receptor guanine nucleotide exchange factor, binds to and activates guanine nu-cleotide binding protein (G-protein) alpha subunit GNAI3 down
KRT4 Keratin, type I cytoskeletal 4 Epithelial keratin gene family Specifically expressed in differentiated layers of mucosal and esophageal epithelia down
CXCL12 C-X-C Motif Chemokine Ligand 12 Physiological processes, tumor related, immune cells, ion channels, inflammation stromal cell-derived alpha chemokine member of intercrone family Protein functions as ligand for G-protein coupled receptor, chemokine (C-X-C motif) receptor 4; CXCR4 activated to induce rapid and transient rise in level of intracellular calcium ions and chemotaxis. Plays roles in embryogenesis, immune surveillance, inflammation response, tissue homeo-stasis, tumor growth/metastasis; che-moattractant active on T-lymphocytes and monocytes but not neutrophils, stimulates migration; several critical functions in embryonic development, bone marrow and heart ventricular septum formation, B-cells down
1 2 3 4 5 6
DIO2 Iodothyro-nine Deiodin-ase 2 Tumor related iodothyronine deio-dinase family Protein is selenoprotein w non-standard amino acid Sec, which encoded by the UGA codon that signals translation termination down
CCL21 C-C Motif Chemokine Ligand 21 Immune cells, ions, inflammation CC cytokines genes Immunoregulatory and inflammatory processes; encoded protein inhibits hemopoiesis and stimulates chemo-taxis; chemotactic in vitro for thymocytes and activated T-cells, not for B cells macrophages or neutrophils; cytokine also plays role in mediating homing of lymphocytes to secondary lymphoid organs down
GNA14 G Protein Subunit Alpha 14 Signaling guanine nucleotide binding/ G protein family Modulators/transducers in various transmembrane signaling systems down
GCNT3 Glucosaminyl (N-Acetyl) Transferase 3, Mucin Type Antigens N-acetylglucosaminyl-transferase family Introduce the blood group I antigen during embryonic development down
BOC BOC Cell Adhesion Associated, Oncogene Regulated Signaling immunoglobulin/ fibronectin type III repeat family Cell-surface receptor com-led that mediates cell-cell interactions between muscle precursor cells, promotes myogenic differentiation down
PLAC8 Placenta Associated 8 Tumor related cornifelin family Might enable chromatin binding activity, positive regulation of cold-induced thermogenesis, positive regulation of transcription by RNA polymerase II, acts upstream/within several processes (brown fat cell differentiation, defense response to bacterium, response to cold) down
GULP1 GULP PTB Domain Containing Engulfment Adaptor 1 Tumor related nucleocytoplasmic shuttling protein Protein encoded is adapter protein necessary for engulfment of apoptotic cells by phagocytes; modulates cellular glycosphingolipid and cholesterol transport; may play role in internalization and endosomal trafficking of various LRP1 ligands such as PSAP down
4. Discussion
According to the resulting table, the most prevalent function of the differentially expressed genes is the regulation of physiological processes. However, as physiological processes is a generally broad topic, it is necessary to take the more detailed description into account. Most notably, there are 5 genes that are used in embryonic development: GJA1, HOXDIO, LAMB3, MMP10, CXCL12. The mutation of genes that are responsible for embryonic development causes the increased potential for developing cancer, as the inability of embryonic cells to develop proper structures that regulate important functions may encourage cancer development. It is connected to hereditary cancer and the tendency for certain groups of demographics to develop cancer.
Secondarily, the tumor-related genes. Again, tumor-related is a broad topic, and detailed descriptions are needed; however, the functions of these genes can be easily connected to the reasons behind recurrence and tumor development. As expected, all genes in this section are downregulated - the disappearance of these genes will increase the likelihood of cancer recurring. Thus, this paper will not be discussing their functions in detail.
Third, inflammation. In the inflammation section, there is only one gene that is upregulated: CXCL1. The rest, HPGD, SCGB1A1, CXCL12, and CCL21, are all downregulated. Inflammation is the body's response to tissue damage, which can be caused by physical injury, infection, exposure to toxins, or other types of trauma. Inflammation causes the repairing of damaged tissue and cellular proliferation. If the cause persists or certain control mechanisms fail, inflammation can become chronic. Once this occurs, tissue repair and cell proliferation will often create an environment in which cancers have a tendency to develop [4]. This information supports the notion that the differentially expressed genes in this table are connected to recurrence. If those that usually regulate the inflammatory response shut-down, mutate, and do not occur frequently in the tumor sites, the
inflammatory responses will become chronic and cancer will develop again, even if it is removed.
Fourth, ion regulation. Studies show that increases in intracellular calcium may inhibit apopto-sis, depending on concentration level, location, and timing [5]. In the results above, all 4 genes, TTYH3, TENM2, NUCB2, and CXCL12, are connected to the calcium ion. Two are upregulated, two are downregulated, respectively. TTYH3 encodes for a calcium channel; if upregulated, it therefore increases the calcium ion channels in the cell membrane, which may cause an increase in the intracellular calcium. Using an intracellular calcium source, TENM2 is involved in calcium-mediated signaling. The upregula-tion of this gene will therefore cause an influx of calcium into the cell. NUCB2 is responsible for calcium level homeostasis. Downregulation of it will cause a destruction of balance; an increase in intracellular calcium will not be returned to normal. CXCL12 activates CXCR4, which induces a rapid increase of calcium levels inside the cell. All these causes added together increase the possibility of the intracellular level of calcium reaching the point of inhibition of apoptosis. Due to this inhibition, the likelihood of tumor development increases; recurrency can occur.
Fifth, cell surface adhesion. All cell surface adhesion genes are upregulated in recurrent tumors. It is said that cell surface proteins are capable of restricting cell growth through contact inhibition; alterations of these molecules are common in cancer [6]. CAM-DR (Cell adhesion mediated drug resistance) is a significant limitation to the success of cancer therapies, most notably chemotherapy. The explanation to why this occurs can be explained by the FN model, which shows the cellular arrest in the G1 phase of cellular division, which significantly reduces the efficacy of drugs [7]. Thus, the upregulation of cellular adhesion molecules may inhibit the initial development of cancer to a certain extent; however, once cancer develops, it negatively impacts the efficiency of treatment, which explains why it is prevalent in recurrent tumor cells.
5. Conclusion
To summarize, most genetic mutations cause differential gene expressions in genes regulating embryonic development, tumor-regulation, inflammation, intracellular calcium level regulation, and cell surface adhesion molecules. Mutation in these functions are proved to be responsible for cancer development or secondary cancer development. However, the upreg-ulation and downregulation of these genes appears unconnected to HNSCC specifically; instead, they seem more connected to cancers in general. These results are still relevant to the objective of the pa-
per- the understanding of the mechanisms in cancer recurrence is helpful in HNSCC recurrence identification, and potential future treatments. Additionally, since recurrence is connected to survival rates, by looking at the genes in the tumor samples doctors will be able to predict the patients' survival rates after treatment.
Acknowledgments
I would like to thank Dr. Pingzhang Wang for introducing to me the tools used in this study, answering my questions when I was confused, and guiding me to my understanding of this topic.
References:
1. Chang Wu, Yuan T. H. Wu and Wu. Locoregionally recurrent head and neck squamous cell carcinoma: incidence, survival, prognostic factors, and treatment outcomes, NIH 2017.
2. Johnson E., Burtness, C. Leemans, Lui, E. Bauman, and R. Grandis, Head and neck squamous cell carcinoma, NIH 2020.
3. Head and neck squamous cell carcinoma, Medicine Plus 2015.
4. Singh, Baby, Rajguru, Patil, Thakkannavar, Pujari, Inflammation and Cancer, NIH 2019.
5. Fnu, Weber, Alterations of Ion Homeostasis in Cancer Metastasis: Implications for Treatment, NIH 2021.
6. Moh, Shen. The roles of cell adhesion molecules in tumor suppression and cell migration, NIH 2009.
7. Huang, Wang, Tang, Qin, Shen, He, Ju. CAM-DR: Mechanisms, Roles and Clinical Application in Tumors, Frontiers 2021.
