Pioneering Clinical Investigation: Beta Defensin Expression in Patients of Squamous Cell Carcinoma of Oral Cavity as a Forward-Looking Validation for Molecular Treatment

Abstract

Human defensins are peptides coded by certain genes released as a part of immune response that act differently to tumorigenic cells according to the class. A varied cellular response has been documented in the world literature concerning the role of defensins in the cancer cell lines, though the exact role in the internal human milieu and the role specific to head and neck cancers is still understudied. This study explores the possible role of Human Beta Defensin (HBD) 1 and 3 in head and neck squamous cell carcinoma in disease progression and their potential as biomarkers/prognostic markers. Two samples, one from the cancerous tissue and one from the normal tissue margin were taken and sent for molecular analysis, viz. mRNA isolation and PCR. The fold change gene expression of HBD 1 and 3 was calculated using a housekeeping gene, and then, as per the available results, we tried to objectify the relative expression of HBD 3 and HBD 1 and its ratio. Out of the 40 samples, 26 were adequate and processed. Expression of HBD1 (DEFB1) and HBD3 (DEFB-103 A) was assessed. Normal tissues were taken as control. GADPH (Glyceraldehyde-3-Phosphate dehydrogenase) was used as an internal control. The cycle of quantification (cq) was calculated for the cases and control. A more than two-fold (2.85 times) increase in expression of HBD1 was found; however, no expression of HBD3 was shown in both cancer tissue and control tissue samples. Additional investigations with a larger sample size, encompassing the expression analysis of cancer genes at various clinico-histopathological stages, are imperative before designating them as biomarkers.

Introduction

Human defensins are peptides coded by specific genes released as a part of the immune response [1], which act differently to tumorigenic cells according to the class [2]. Human defensins are classified into alfa and beta classes. Alfa class of human defensin are activated from lymphocytes and intestinal Paneth cells. Alfa defensins at various concentrations show antimicrobial and tumoricidal effects. The beta class of defensins are more pronounced in mucosal and epithelial cells. Hence, the possibility of beta-defensin expression in head and neck carcinoma [3] and its quantification by various techniques was studied. The most studied beta-defensins are HBD1 and HBD3 [1]. Overexpression of HBD3 [4] in neoplastic cells, including dysplastic cells, suggests an oncoprotein effect compared to normal tissue, which shows minimal expression of HBD3, while HBD1 acts as a tumour suppressor protein. Hence, downregulation of the same is seen in tumour cells. The Alfa class shows expression in both inflammatory and tumorigenic cells, and the beta class is more specific for tumour cells. Hence, studies regarding beta-defensins are crucial for establishing their relation as a tumour marker, prognostic index, and targeted therapy.

There are varied cellular responses documented in the literature concerning the role of defensins in cancer cell lines. However, there needs to be more literature regarding its exact role in the internal human milieu and the role specific to head and neck cancers. Previously, in the literature, SELDI-TOF-MS on IMAC30 Protein-Chip Arrays showed various aberrations in defensin expression [5] in healthy mucosa compared to tumour cell lines. Similarly, evidence in the literature shows an increased proliferation of TR 146 cancer cell lines in Petri dishes with the administration of human beta-defensin 3 with VEGF inhibiting cell migration. In some tumour cell lines, the human beta-defensin 1 [6] is downregulated. Joly S et al. [7] showed differential expression of HBD 3 and consistently low expression of HBD 1 and 2 in oral squamous cell lines compared to controlled cell lines.

Kawsar HI [8] et al. showed overexpression of the HBD 3 gene in oral dysplastic lesions but not of the HBD 1 and 2. Their in-vitro studies showed the chemoattractant nature of HBD 3 on THP 1 monocytic cells and a significant increase in HBD 3 levels by epidermal growth factor by MAP kinase pathways such as of MEK ½, p38 MAPk, protein kinase C and PI3K cellular signalling pathways, interestingly except Janus kinase (JAK) pathway. Through an in vitro study on cell lines, they could prove the role of HBD-3 as a mitogen-responsive gene for oral cancer initiation and motility signal to recruit tumour-associated macrophages. Das Gupta T proposed a pathway for HBD3 expression via the p53 by HPV oncoprotein E6 [9]. Hence, there is a possible role of HBD 1 and 3 in the progression of human cancers, such as head and neck cancers and in behaving as a biomarker/potential prognostic marker.

Considering field cancerization, evidence of differential expression of HBD 3 in cancer cell lines with predominant overexpression [10], including dysplastic cell samples and the in-vitro role of HBD 3 in the tumour progression with consistent downregulation of HBD 1 in the squamous cell lines, we propose to study the relative expression of HBD 3 and HBD 1 in head and neck cancer tissue samples relative to normal margin controls. We also propose to study the proportion of HBD 3/ HBD 1 as a possible prognostic marker for clinical tumour behaviour.

Objectives

To study expression of HBD 3 and HBD 1.

1. Objectify the relative expression of HBD 3 and HBD 1.

2. Proportion of HBD 3/ HBD 1 as a possible prognostic marker for determining clinical behavior.

Methodology

Study type

Experimental study - laboratory based was conducted as a part of pintramural research project in our tertiary care institute.

Inclusion Criteria

Patients of head and neck squamous cell carcinoma.

Exclusion Criteria

Patients with known mutation diagnosed tumour syndromes of pre confirmed familial cancer syndromes were excluded as their molecular progression is already established. Patients not consenting for the study were also excluded.

Methods: Consent was taken in both English and in native language from all the subjects. Post recruitment and consent, patients were taken for definitive surgery as per the routine departmental protocol and two punch samples, one each from the cancerous tissue (Carcinoma of Buccal mucosa) and normal tissue margin were taken intraoperatively and sent for analysis in the molecular biology laboratory as per the following:

Data Analysis

The fold change gene expression of HBD 1 and 3 was calculated using house-keeping gene and then as per the available results, we have tried to objectify the relative expression of HBD 3 and HBD 1 and its ratio.

AJCC 8th staging was used in all the patients. Staging for buccal mucosa was used which involves the subsites Mucosa of lips, Cheek and retro molar areas, vestibule of mouth, alveolus, gingiva and hard palate and tongue (See Table 1).

Table 1.

Baseline study characteristics

Serial Number	Age	Clinical staging	Clinical stage AJCC 8th	Pathological staging	Smoking If yes (YEARS)	Tobacco Chewing If yes (YEARS)	Alcohol If yes (years)	∆∆ct	Fold Change expression of HBD1
1	51	T4aN3bM0	IVB	pT4aN3b	No	20	No	-1.14	2.20
2	53	T4aN2bM0	IVA	pT4aN3b	No	15	No	2.50	0.18
3	49	T4aN2bMx	IVA	pT4aN2b	No	10	No	-7.82	225.34
4	72	T4aN0M0	IVA	pT4aN0	30	No	No	-1.68	3.20
5	59	T4aN0M0	IVA	pT4aN1	No	15	No	-5.33	40.31
6	61	T4aN0M0	IVA	pT4aN0	No	20	No	-5.89	59.26
7	69	T4bN2bM0	IVB	pT4aN2b	30	30	No	2.00	0.25
8	57	T4bN3bM0	IVB	pT4aN2b	25	20	No	3.53	0.09
9	61	T4aN2aM0	IVA	pT4aN2a	20	No	No	-2.58	5.99
10	53	T4aN2bM0	IVA	pT4aN2b	No	20	No	-2.52	5.72
11	72	T4aN3bM0	IVB	pT4aN3b	20	50	No	8.04	0.00
12	42	T4aNoMo	IVA	pT4aN0	15	No	No	2.68	0.16
13	67	T4bN3bM0	IVB	pT4bN3b	15	20	No	4.21	0.05
14	66	T4aN0M0	IVA	pT4aN1	20	10	No	-0.28	1.21
15	60	T3N1M0	III	pT3aN1	No	12	No	-1.91	3.76
16	66	T3N2aM0	IVA	pT3N2a	30	No	No	1.79	0.29
17	60	T4aN0M0	IVA	pT4aN1	20	No	No	-2.32	5.00
18	62	T3N0M0	III	pT2N2b	No	No	No	-0.93	1.90
19	43	T4aN0M0	IVA	pT4aN0	No	15	No	-1.82	3.52
20	33	T4aN1M0	IVA	pT2N2a	N0	12	2	1.17	0.44
21	42	T3N0M0	III	pT3aN0	No	15	No	-5.89	59.28
22	43	T4aN0M0	IVA	pT4aN0	5	1	No	-4.57	23.79
23	62	T4aN3bM0	IVB	pT4aN3b	No	15	No	-4.70	25.92
24	68	T3N0M0	III	pT2N0	30	No	No	-2.66	6.33
25	75	T4aN1M0	IVA	- pT4aN0	No	10	No	-1.96	3.90
26	53	T3N0M0	III	PT2N0	N0	20	N0	-11.21	2363.57

Table showing the details of the patient participants who underwent surgery, including their history of addiction

Results

Out of the 40 samples 26 samples were adequate and processed for RT-PCR. Expression of HBD1 (DEFB1) and HBD3 (DEFB-103 A) was assessed. Normal tissues were taken as control. GADPH was used as internal control. Cycle of quantification (cq) was calculated for the cases and control.

∆ct is the difference between cases or control (Sequence of interest, Cq) and reference sequence internal control (GADPH)(Cq) [11].

∆∆ct (difference in ∆ct of cases and controls) from which final fold change expression was found.

A mean of than two fold increase of expression (2.85) of HBD1 was found, however no expression of HBD3 was shown by either cases or controls.

Clinico-pathological Correlation

Post Hoc Analysis (one way Anova) with fold change variability of HBD1 for different clinical stage of cancer. (ANOVA p < 0.001) (Table 2).

Table 2.

Fold change expression of HBD1 in different clinical stages of the disease

Fold Change expression of HBD1
Tukey Ba, b
TUMOUR CLINICAL STAGE(AJCC8TH)	N	Subset for alpha = 0.05(*)
		1
IV B	6	4.7517
IV A	15	25.2207
III	5	486.9680

• Stage III cancer have high fold change increase of 486.96 times (~ 500 times).

• Stage IVA and IV B have less fold change variability of 25.22 and 4.75 times respectively (< 100 times).

Post Hoc Analysis (one way Anova) with fold change variability of HBD1 for different clinical stage of cancer. (ANOVA p < 0.001)

Discussion

There is evidence in the literature regarding the possible differential expression of human beta-defensin, HBD 3 and under-expression of HBD 1 in cancer cell lines and in biopsies from dysplastic lesions. There are possible mechanisms described in the literature regarding the role of HBD in human cancer progression. However, there is no clinical evidence regarding the possible expression in human cancer biopsies, specifically in head and neck cancers. We proposed addressing this lacuna by studying human head and neck cancers regarding HBD 3 and HBD 1 expression. Multiple in vitro studies have been conducted on different cell lines, including cancer precursor cells, showing a variable expression of HBD3, upregulation and acting like a mitogen-responsive gene and activating oncoproteins. The expression of HBD1 is not consistent in the available literature. The HBD1 gene usually functions by expressing tumour suppressor protein. Downregulation of HBD1 tissue was noted in various carcinomas, especially renal cell carcinoma. The downregulation of the HBD1 gene by its hypermethylation has been proven in oral cancers. Joly S et al. showed differential expression of HBD 3 and consistently low expression of HBD 1 and 2 in oral squamous cell lines compared to controlled cell lines. Kawsar HI [8] et al. showed overexpression of the HBD 3 gene in oral dysplastic lesions but not the HBD 1 and 2.

We studied the expression of HBD 3 and 1 in head and neck cancer tissue samples, comparing normal margin controls in the feasible locations. We also propose the HBD 3/ HBD 1 ratio as a possible prognostic marker for clinical behaviour. The study was done as a pilot project with a sample size decided as per feasibility because of the absence of literature concerning the same previously. Cases with known mutations in cancer syndromes, cases of known familial cancer syndromes, and those not consenting to the study were excluded. Out of 40 intra-operative head and neck squamous cell carcinoma samples, 24 were suitable for final analysis and subjected to the final PCR.

Observations revealed a tendency for cancer patients skewed toward later prognostic stages. Notably, the post hoc analysis results were statistically significant (p-value < 0.05), suggesting that the gene’s expression could serve as a marker. Our investigation uncovered diminished tumour suppressor gene expression in the advanced stages of the disease, indicating a poorer prognosis for stage IV B disease. (Table 2, Figure 1) The expression levels at the individual patient level in the early stages of the disease may align with the disease’s aggressiveness and resistance to treatment. A significantly high expression of the tumour suppressor gene was observed in stage III. (Table 2, Figure 1), which could indicate better survival outcomes. However, the study did not include patients in stage I and stage II to observe the behaviour of tumour suppressor gene levels in those stages. In the Indian population, delays [12, 13] in reaching a tertiary care institute lead to late diagnosis and definitive treatment. Hence, most patients presenting to the outpatient clinic are in the late prognostic stage, T4, with multiple nodal metastases. Given this study’s preliminary nature, conducting a more extensive investigation involving a larger and more diverse patient cohort across various stages is recommended. A multicentric study would be beneficial for validation. Additionally, we propose conducting molecular genetic studies to activate HBD1, as this could slow disease progression, leading to a better prognosis and reduced morbidity and mortality. This expanded study could prove valuable in examining the behaviour and expression patterns of different beta-defensins and their association with various addiction patterns.

Fig. 1.

Showing mean of fold chain expression of HBD1 in various stages of carcinoma of buccal mucosa

HBD 1 and 3 in human cancers, such as head and neck cancers in progression, can behave like a biomarker/potential prognostic marker. These two genes’ exact mechanisms and roles have yet to be established. Inconsistency in the expression of these two genes is not known. Most studies, especially in precancerous cells, showed an upregulation of the HBD3 gene. Hence, we expect a similar result in a proven head and neck cancer. Similarly, we expect the downregulation of HBD1 in cancer cells because the existing hypothesis supports that it can function as a tumour suppressor gene in vitro. However, the results were different from the existing literature.

All tumour biopsy samples exhibited the expression of HBD1, revealing a mean 2.85-fold increase compared to normal tissue. Interestingly, our study contradicted existing findings by revealing up-regulation of HBD1 in head and neck squamous cell carcinoma. Moreover, there is currently no evidence indicating the expression pattern of the HBD1 gene in the Indian population.

The role of defensins in tumour development and progression remains contentious, with certain groups reporting the overexpression of specific β defensins in certain cancer types. Notably, elevated levels of HBD1 and HBD2 were detected in the sera of lung cancer patients. Another instance includes the overexpression of HBD1 in renal cell carcinoma and HBD3 in oral squamous cell carcinoma (OSCC) [14, 15]. Our hypothesis posits that the observed upregulation of tumour HBD1 in the Indian population may be associated with a better survival rate, possibly due to the upregulation of tumour suppressor genes or differences in the gene pool (population genetics). Additionally, the absence of HBD3 expression and the overexpression of HBD1 in cancer cells compared to precancerous cells could indicate a negative feedback mechanism at the gene level during different disease stages.

One of our study objectives was to establish a correlation between HBD1 and HBD3. Unfortunately, none of the samples exhibited significant expression of HBD3, preventing the establishment of an index marker using HBD1 and HBD3 expression levels in head and neck tumours. The potential association between human papillomavirus (HPV) gene-related head and neck cancer and the differential expression of the HBD1 gene cannot be ruled out. Although there is currently no evidence supporting a link between the HBD1 gene and HPV virus expression [9], ongoing studies in population genetics may provide novel evidence to support our hypothesis.

Conclusion

Additional investigations with a larger sample size, encompassing the expression analysis of cancer genes at various clinico-histopathological stages, are imperative before designating them as biomarkers. Notably, HBD3 expression was absent in both the control and tumour tissues; consequently, a prognostic index considering HBD3/HBD1 was not computed.

For forthcoming studies, it may be worthwhile to explore the relative expression of cancer genes in diverse ethnic groups, also incorporating correlation with addiction history, as this could provide valuable insights into potential variations and patterns across different populations. This approach could contribute to a more comprehensive understanding of the role of defensins as potential biomarkers in cancer.

Prospects and Directions

It is important to note that our study solely focused on the Indian subset of the population. Consequently, the findings should be separate from a global context. Separate investigations are warranted to explore the prevalence of HPV and the expression of cancer genes in HPV-positive groups, considering the nuances of population genetics.

The correlation between gene expression levels and addiction history can offer valuable insights into the impact of addiction and the severity of the disease. This correlation may provide a new avenue for predicting life expectancy and disease prognosis.

Additionally, future studies should emphasize confirmed malignancies at various clinical and radiological stages. This approach will provide a more nuanced and specific insight into the role of defensins and related factors in different stages of malignancy, contributing to a more robust understanding of their implications in diverse clinical scenarios.

Author Contributions

Vishudh Mohan: Data collection and processing, Analysis, Materials, Writing. Sourabha Kumar Patro: Conception, Design, Literature Review. Purvi Purohit: Critical Review. Vidhu Sharma: Critical Review. Kapil Soni: Critical Review. Bikram Choudhury: Literature Review. Amit Goyal: Supervision, Literature Review.

Declarations

Conflict of Interest

No conflict of interest.