|Ahead of print publication
Stage-specific expression analysis ofMMP-2 & MMP-9 in laryngeal carcinoma
Jaimanti Bakshi1, Atul Kumar Goyal1, Virender Singh1, Malay Sannigrahi2, Madhu Khullar3
1 Department of Otolaryngology and Head Neck Surgery (ENT), PGIMER, Chandigarh, India
2 Department of Chemical Sciences, IISER, Mohali, Punjab, India
3 Department of Experimental Medicine and Biotechnology, PGIMER, Chandigarh, India
Atul Kumar Goyal,
Department of Otolaryngology and Head Neck Surgery (ENT), PGIMER, Chandigarh - 160 012
Source of Support: None, Conflict of Interest: None
Aim of the Study: Both matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) is involved in degradation of extracellular matrix and found to stimulate invasion and metastasis in cancer patients. However, studies on the stage-specific expression of MMPs at different stages of larynx carcinoma are still lacking. In the present study, we compare the expression level of MMP-2 and MMP-9 at different stages of laryngeal carcinoma.
Material and Methods: Tumor tissues samples were taken from larynx cancer patients by deep biopsy during direct laryngoscopy. Gene expression for MMP-2 and MMP-9 was analyzed using RT-PCR.
Results: Significantly high expression of MMP-2 was observed compared to the MMP-9 at stage IV compared to the less advanced stages of the disease.
Conclusion: Present study concluded that the MMP-2 expressed with a greater magnitude as compared to the MMP-9 in advance stages of laryngeal carcinoma.
Keywords: Biopsy, cancer, gene expression, laryngeal, matrix metalloproteinases
| > Introduction|| |
Laryngeal carcinoma is the sixth most common cancer worldwide, constituting 25% of all head and neck malignancies. In India, it is the seventh most common cancer in males , and accounts for approximately 2% of all cancers., Apart from the general symptoms of laryngeal carcinoma including complete airway obstruction, severe respiratory distress, and the inability to speak,,, one of the hallmarks of laryngeal cancer is distant metastasis which decreases survival by almost 50%, and its further invasion beyond the lymph nodes makes the situation worst.,, Matrix metalloproteinases (MMPs) are a collection of enzymes capable of cleaving extracellular matrix components which are a key factor for invasion and metastasis. MMP family is a group of zinc ion-dependent endopeptidases, and at present, 26 members of the MMP family have been identified which are numbered as MMP 1–26. According to their substrates and the homology of the generated fragments, MMPs are divided into six classes: Collagenases, gelatinases, matrilysins, stromelysins, furin-activated MMPs, and other secretory MMPs. Among all MMPs, type IV collagenase (MMP-2 and MMP-9) plays a pivotal role in carcinoma  as it degrades the type IV collagen, which is a major component of basal membrane.
High expression of MMP-2 has been found in human colorectal cancer, gastric carcinoma, breast cancer, lung cancer, prostatic carcinoma, and head and neck cancer. Similarly, high expression of MMP-9 was found to be related to lower survival of patients with lung cancer, rectal carcinoma, and esophageal carcinoma. Moreover, it was associated with the severity of tumor invasion, lymphatic metastasis, venous tumor thrombus, and pathological stages. Both MMP-2 and MMP-9 were also found to be highly expressed in laryngeal cancer,,, but a comparative expression of MMP-2 and MMP-9 in laryngeal cancer is still controversial. Some studies demonstrated that MMP-9 is overexpressed in laryngeal cancer, while other studies showed overexpression of MMP-2., Moreover, expression data with respect to specific stage of laryngeal cancer are still lacking.
A significant amount of effort has been made into the development of MMP inhibitors, and synthetic inhibitors have been developed as a new class of potential anticancer drugs in recent years. Although observations of MMP inhibitors in mouse models demonstrated promising results, clinical trials have been uniformly disappointing. Because MMP family has many enzymes, it is important to find out particular target protein against which new drugs can be synthesized. Therefore, in the present study, we studied the expression of MMP-2 and MMP-9 at the gene level and compared the respective magnitude of expression for both of them, so that the potential target enzyme can be defined. Moreover, we also analyze the cancer stage-specific expression of MMPs, so that target cancer stage could also be identified. The present study will help in defining specific role of each MMP in laryngeal cancer in a stage-specific manner, so that further research on that could be conducted to discover specified MMP inhibitors.
| > Materials and Methods|| |
The study comprised 15 laryngeal cancer patients who visited the Otolaryngology and Head and Neck Surgery (ear, nose, and throat [ENT]), Outpatient Department (OPD), PGIMER, Chandigarh, between 2017 and 2018. Patients having an age range between 20 and 60 years were taken in the study.
A small piece of tissue was cut from deep inside the tumor in the larynx. All biopsies were performed in a minor operating room of ENT, OPD, by doing direct laryngoscopy. Local anesthesia was administered to prevent pain, which may occur during the biopsy.
Total RNA and isolation
Total RNA was obtained from the tissue sample stored at −80°C in RNA stabilizing agent, RNAlater. RNA was extracted using a commercial kit available for the purpose of using manufacturer's protocol. RNA concentration was measured using an ultraviolet-visible spectrophotometer by taking absorbance at 260 nm (A260) and an optical density. RNA was then kept at −80°C until use.
Complementary DNA synthesis
About 500 ng–5 μg total RNA was used to synthesize the complementary DNA (cDNA). Commercially available kits for the purpose were used to synthesize the cDNA following manufacturer's protocol. Obtained cDNA was then kept at 4°C until use.
Real-time polymerase chain reaction
Changes in gene level expression were detected by real-time polymerase chain reaction (RT-PCR). An equal amount of cDNA was amplified using MMP-2 primers (F: 5'-CTCATCGCAGATGCCTGGAA-3', R: 5'-TTCAGGTAATAGGCACCCTTGAAGA-3'), and MMP-9 primers (5'-GATGCGTGGAGAGTCGAAAT-3', 3'-CACCAAACTGGATGACGATG-5'). Amplified products were quantitated in terms of 2^-ΔCT.
The statistical analysis was carried out using GraphPad Prism Software version 7.04 (GraphPad Software Inc., San Diego, CA). The mean and standard deviation were calculated. The Student's t-test was used to compare the means, and P < 0.005 was considered as statistically significant. Two-Way ANOVA was used for data analysis.
Language, grammar, and plagiarism
The references were inserted using EndNote software version 7.4 (Thomson Reuters, Toronto, Canada), language and grammar was checked by Grammarly software version 6.6 (Grammarly, Inc., San Francisco, California, United States), plagiarism was checked with the help of Turnitin plagiarism detection service (Webster St., California), proofread by doc navigator ©, Chandigarh.
| > Results|| |
Four samples out of 15 do not give the sufficient yield of RNA and cDNA, which were required for RT-PCR, and therefore, complete expression analysis was carried out for 11 samples. Both MMP-2 and MMP-9 do not show any expression in tissue samples of Stage I laryngeal carcinoma. Tissue samples of Stage II and III show mild expression for MMP-2 and MMP-9. The highest expression for MMP-2 and MMP-9 was observed in Stage IV tissue samples. Overall, the MMP-2 expression is markedly high compared to MMP-9 for all carcinoma stages [Figure 1] and [Table 1].
|Figure 1: Expression level of MMP-2 and MMP-9 in terms of 2^-ΔCT at different stages of larynx carcinoma|
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|Table 1: Mean expression values of MMP-2 and MMP-9 in terms of 2^-ΔCT at different stages of larynx carcinoma|
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| > Discussion|| |
Metalloproteinases are responsible for a continuous process of remodeling the extracellular matrix structure and its correct composition. Their main function is to digest the components of the extracellular matrix and the basement membrane of the vessel, facilitating tumor growth, cell migration, and tumor invasion, leading to metastasis. Increased expression of MMP-2 in the tumor tissue is correlated with clinical stage, metastases, recurrence and survival. The 5-year survival rate in laryngeal cancer has remained unchanged to a large extent over the past 30 years. This trend is attributed mainly to recurrent neck disease for which few biological markers have been described. Therefore, we attempted to evaluate the potency of MMP-2 and MMP-9 as a prognostic marker of survival and neck disease in a group of patients with squamous cell carcinoma of the larynx.
MMPs play a key role in several steps of tumor progression, which includes the angiogenesis and metastasis. In some tissues including larynx, it is demonstrated that the MMPs have an additional enzymatic activity that may affect the cancer invasion. Previous studies have tried to show the role of MMPs in the cancer progression and metastasis in different cancers., The serum levels of MMP-2 and MMP-9 were found to be significantly increased in laryngeal squamous cell carcinoma (SCC) compared to normal healthy controls. Similarly, MMP-2 and MMP-9 were also found to be overexpressed in laryngeal carcinoma tissue, in an earlier study conducted using immunohistochemistry., However, unlike these studies which have evaluated the expression of these markers in serum or immunohistochemically, we evaluated the MMP-2 and MMP-9 expression at gene level while taking cancer-stage specificity in concern.
Although our study revealed the gene level expression of MMP-2 and MMP-9 in laryngeal carcinoma, still the present study has several limitations. In the present study, we analyzed the expression of MMP-2 and MMP-9 preoperatively only because the tumor has removed during the surgery. In addition, a sample size of our study is small due to complications with disease and patient's cooperation toward study procedures.
| > Conclusion|| |
Our study gives the proof for the gene level expression of MMP-2 and MMP-9 in a stage-specific manner in laryngeal cancer. The present study will help bridge the existing gap between in vitro and in vivo studies. Knowledge generated from the present study will help in the development of targeted therapy for laryngeal carcinoma, which could be proved useful for routine clinical practice.
Compliance with ethical standards
All procedures performed during the present study were in accordance with the ethical standards of the institutional ethics committee (IEC). The questionnaire and the study procedures have been approved by the IEC. Written informed consent was obtained from all individual participants included in the study.
The authors are highly acknowledged by all the patients who participated in the study.
Financial support and sponsorship
The work in this project is supported by PGIMER Intramural Research Grant (Sanction No. 71/8-Edu-15) and Junior Research Fellowship (JRF) from ICMR, New Delhi (Sanction No. 3/13/JRF -2015/HRD).
Conflicts of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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