Expression Proling of Tumorigenesis Genes in Patients of Laryngeal Papillomatosis

Recurrent Laryngeal papillomatosis (RLP) is a disease of the aero-digestive junction superimposed at the upper respiratory tract. Low risk human papillomavirus type 6 and 11 are infectious agents in RLP. Mainstay of treatment for RLP is CO2 laser vaporization for debulking of papilloma. Understanding of the gene changes at expression level might prove helpful for RLP management. The study was designed to evaluate the changes in tumorigenesis genes in RLP cases to mark the severity and aggression at gene expression level for disease management. Tissue samples and peripheral blood samples were acquired from clinically diagnosed RLP patients of age < 12 years undergoing CO2 laser vaporization. Control samples were taken from the normal thyroid tissue of individuals undergoing thyroidectomy. Relative quantitation of gene expression was done by real time polymerase reaction using cDNA samples. Up regulation of CXCL12/ CXCR4 signaling axis, parallel increase in GLUT-1 and over expression of IGF-I, EGFR and ER-α gene product was observed in RLP. On the other hand HIF-1a levels not discerned elevated along with VEGF, PIK3 and AKT gene expression prole. The expression analysis has provoked the possible strategies which can be planned to treat RLP by adjuvant and tailored medication to reduce multiple surgeries. CXCR4, GLUT-1, IGF-1, HIF-1a, VEGF, ERK1/2, PIK3 and AKT. GAPDH gene expression was determined for relative quantitation of target genes. RT-PCR was done using all genes mRNA specic primers, Maxima SYBR Green/ROX qPCR Master Mix (Thermo scientic, Lithuania) and cDNA in standard cycling program for SYBR Green on RT-PCR Machine (Sansure, China).


Introduction
Patients suffering from this disease develop multiple benign papilloma of the respiratory tract that have a particular predilection for the true vocal cord. On the other hand malignant progression is rare, it appears to be more common in those papilloma harboring high-risk (HR) HPV subtypes. The course of human papilloma virus (HPV)-induced recurrent laryngeal papillomatosis is variable and unpredictable. Some patients experience spontaneous remission after one or two surgical procedure while others suffer from recurrent aggressive growths with dire consequences.
The intensity of nuclear staining of TLR4 was signi cantly lower in laryngeal papillomatosis transforming into laryngeal squamous cell carcinoma (LSCC) as compared to Laryngeal Papillomatosis have not been malignantly transformed (1).
HPV-6 viral load in adult-onset laryngeal papilloma decreased gradually to zero following several surgeries and intralesional cidofovir therapy. These ndings provide evidence that relapses can occur if latent laryngeal HPV reservoirs are not eradicated and HPV replication might re-initiate from those sites after the end of therapy (2).
The human physiology is ardent to respond against or pro benign tumors. Similarly in context of RLP, spontaneous molecular changes may be involved in neoplasia. Therefore, underlying genetic mutations at individual level are contributory in the onset and progression of the disease. A mutation from C to T at codon 273 of the P53 gene at CpG dinucleotide was associated with the integration of HPV-11 in histologically malignant lesions in a 28 year old symptomatic papillomatosis patient (3).
As a consequence of the laryngeal papillomatosis, different signaling pathways are deregulated. Therefore, deregulated underlying molecular mechanisms were studied in the clinically diagnosed patients in the present study. Expression levels of different tumor suppressors and oncogenes were investigated to understand if overexpression or under expression of target genes modulated the disease severity.

Materials And Methods
Subjects: The study was approved for use of tissue biopsy sample by the ethical review committee, KRL Hospital, Islamabad, Pakistan. Experimental protocols were approved by Institute of Biomedical and Genetic Engineering, Islamabad, Pakistan. All the methods were done in accordance with relevant regulations and guidelines. Patients, aged < 12 years presenting to ENT department of tertiary care hospital with respiratory distress and airway blockage due to RLP were requested to participate in this study. Patients Expression pro ling of Tumorigenesis Genes: Genetic expression pro ling was carried out for target genes EGFR, ER-α, CXCL12, CXCR4, GLUT-1, IGF-1, HIF-1a, VEGF, ERK1/2, PIK3 and AKT. GAPDH gene expression was determined for relative quantitation of target genes. RT-PCR was done using all genes mRNA speci c primers, Maxima SYBR Green/ROX qPCR Master Mix (Thermo scienti c, Lithuania) and cDNA in standard cycling program for SYBR Green on SLAN96 RT-PCR Machine (Sansure, China).
A fold change in expression of the target genes in disease relative to normal tissue was calculated. The calculation was done by adjusting the GAPDH expression level (as house-keeping) for target gene expression in disease and normal tissues using double delta cT method.

Results And Discussion
Patients suffering from RLP belong to age less than 10 years. Laryngeal obstruction due to extensive growth in larynx and in some cases trachea and hypopharynx was caused respiratory distress and swallow di culty. The RLP obstruction was removed with CO2 laser ionization under general anesthesia. Expression pro ling for 12 genes along with the house keeping gene was quantitated. The relative fold gene expression analysis between the disease and normal tissue showed following discrete post translational gene expression changes.

i) Epidermal growth factor receptor (EGFR)
Association of Epidermal growth factor receptor (EGFR) has been well established in many tumors. Likewise EGFR translational availability has been implicated in patients of this study. EGFR inhibition in RRP with EGFR inhibitors as an adjuvant therapy has proven to lower RRP operative frequency and improvement in the modi ed Derkay scores and general disease (4) ii) Estrogen Receptor Alpha (ER-α) About 67% patients showed overexpression of Estrogen Receptor alpha (ER-α) which shows their ER positivity. Estrogen Receptors are hormone activated transcription factors with an important role in carcinogenesis (5). ERα-positive cases are not only responsive to endocrine therapies, but also sensitive to CDK4/6 inhibitors (6, 7). ERα-negative tumors, on the other hand, are more aggressive and metastatic (8). Treatment choices of ER positive cases by reported evidence for other cancers or malignancies can be opted for laryngeal papillomatosis.

iii) Chemokine Receptor and its Ligand (CXCL12/CXCR4)
Overexpression of chemokine CXCL12/CXCR4 signaling axis was observed. This favors virus production and HPV induced proliferation. This axis has been veri ed by gain of function mutation in CXCR4. Expression of CXCR4 promoted stabilization of HPV oncoproteins thus disturbing cell cycle progression and proliferation at the expense of the ordered expression of the viral genes required for virus production (9). iv) Glucose transporter 1 (GLUT-1) & Insulin-like growth factor 1 (IGF-1) High grade neoplasm observed in majority of RLP cases in the present study. An increased metabolite uptake by the proliferating cells is demanded, and tumor cells need to improve the nutrient uptake from the environment/serum. Glucose up-take may be regarded is the rate limiting step in neoplastic growth and tumor cell metabolism. Since glucose does not merely drawn across the lipid-bilayer, three specialized categories of cell membrane protein transporters for sugar have been identi ed in mammals as Glucose Transporter 1 (GLUT-1), Sodium-sugar linked Transporters (SGLT) and Sugars will eventually be exported transporter (SWEET) (10,11,12,13). Among these GLUT-1 facilitates glucose transport the concentration gradient dependent glucose transport without requiring the primary or secondary ATP hydrolysis (14). In our study group up-regulation of the GLUT1 gene has been noted in 90 percent of patients. GLUT-1 overexpression can be considered as a strategy to increase glucose uptake for unprecedented cell growth like as other cancer cells hallmark feature. As there has been a parallel increase in IGF-I has been seen in present study. Mechnism of GLUT1-mediated glucose uptake has also been documented in the adrenomedullary cells. And an enhanced glucose transport in response to IGF-I associated activation of IGF receptor type 1 and GLUT1 translocation (15). Enhanced angiogenesis through HIF-1a dependent VEGF expression is hallmark of tumor vascularization and growth. In context of the high-risk HPV type 16; E6 and E7 oncoproteins induced an increase in accumulation of HIF-1a protein and subsequently Hif1-a triggered VEGF expression by ERK1/2 and PIK3/AKT. These were suggestive ndings observed for increased Hif1-α and VEGF expression in tumor growth of cervical cancer (16). E6 and E7 oncogenes also inhibit tumor suppressor p53 and Rb (17). The RLP patient's expression pro ling presented a relatively surprising data in case of Hif1a, VEGF, AKT and PIK3 in the present study. Only in about 2 percent of patients the said genes were overexpressed while the rest of the cases did not show any aberrant expression.
The RLP patient papillomas cause airway obstruction, which leads to fall in oxygen fall in blood oxygen levels in spontaneously breathing patient. In this event patient also retain CO 2 so that their blood CO 2 rises. Hypoxia (low oxygen) and hypercapnia (high carbon dioxide) are concurrently present in tissue microenvironment in variety of pathophysiological conditions due to respiratory diseases e.g. obstructive sleep apnea syndrome, pneumonia and chronic obstructive pulmonary disease (COPD) (18, 19). The hypercapnia has been well studied in vivo and in vitro to counter-regulate and suppress hypoxia induced Hif1-α pathway activation. The mechanism involves CO2 dependent pH reduction which assists noncanonical lysosomal degradation of Hif1-α protein (20). This can be best hypothetical model for the pathophysiology for current study where Hif1-α and its target genes expression was not overexpressed in presence of high CO 2 retention in airway obstructed patients.

Conclusion
The extract of the study highlights important molecular based targeting mechanisms for RLP and to put a barrier in papillomatosis development. The key targets as understood in the genetic expression pro ling include; Inhibition of chemokine receptors through targeted therapy, adjuvant therapy by EGFR inhibitors, ER α antagonist Tamoxifen, which is structurally similar to estrogen is used for patients with ER α over expression. And therapies for inhibition of Hif1-α may not prove e cient in this disease. A quick expression pro ling for cell proliferation and oncogenes in RLP patients can reveal the molecular targets to treat with available and or adjuvant therapy. Furthermore antivirals still stand to compensate the disease. Further insights at the level of protein analysis may facilitate new treatment regime formulation.

Con ict of Interest
The authors did not declare any con ict of interest.