Differential transmission of the molecular signature of RBSP3, LIMD1 and CDC25A in basal/ parabasal versus spinous of normal epithelium during head and neck tumorigenesis: A mechanistic study

<div><p>Head and neck squamous cell carcinoma (HNSCC) is a global disease and mortality burden, necessitating the elucidation of its molecular progression for effective disease management. The study aims to understand the molecular profile of three candidate cell cycle regulatory genes, RBSP3, LIMD1 and CDC25A in the basal/ parabasal versus spinous layer of normal oral epithelium and during head and neck tumorigenesis. Immunohistochemical expression and promoter methylation was used to determine the molecular signature in normal oral epithelium. The mechanism of alteration transmission of this profile during tumorigenesis was then explored through additional deletion and mutation in HPV/ tobacco etiological groups, followed byclinico-pathological correlation. In basal/parabasal layer, the molecular signature of the genes was low protein expression/ high promoter methylation of RBSP3, high expression/ low methylation of LIMD1 and high expression of CDC25A. Dysplastic epithelium maintained the signature of RBSP3 through high methylation/ additional deletion with loss of the signatures of LIMD1 and CDC25A via deletion/ additional methylation. Similarly, maintenance and / or loss of signature in invasive tumors was by recurrent deletion/ methylation. Thus, differential patterns of alteration of the genes might be pre-requisite for the development of dysplastic and invasive lesions. Etiological factors played a key role in promoting genetic alterations and determining prognosis. Tobacco negative HNSCC patients had significantly lower alterations of LIMD1 and CDC25A, along with better survival among tobacco negative/ HPV positive patients. Our data suggests the necessity for perturbation of normal molecular profile of RBSP3, LIMD1 and CDC25A in conjunction with etiological factors for head and neck tumorigenesis, implying their diagnostic and prognostic significance.</p></div

Overall concordance obtained between different experiments undertaken in invasion tumours.

<p>Abbreviations: EXPR: Expression (IHC); METH: Methylation (promoter); DEL: Deletion; OVERALL: overall alterations (Deletion+ Methylation);DYS: Dysplasia; HNSCC: Head and neck squamous cell carcinoma; H: High; M: Moderate; L: Low; ND: Not done; NOR: Normal; TUM: Tumor; B: Basal; P: Parabasal; S: Spinous. P value represents level of significance.</p

Promoter methylation analysis.

<p>Promoter methylation of the genes in normal epithelium (basal/ parabasal and spinous layers), dysplastic zone of epithelium and HNSCC. U: Unmethylated; M: Methylated; N: Normal; T: Tumor; B: Basal layer; P: Parabasal layer; S: Spinous layers; L: pUC19/ HpaII molecular weight ladder. a. Representative images depicting step- wise separation of basal/parabasal and spinous layers in normal epithelium.i. Hematoxylin and eosin stained parallel normal section. ii. Unstained parallel section. iii. Laser line (arrow star) demarcating basal/ parabasal and spinous layers. iv. Remaining basal/ parabasal layers after separation of the spinous layers. b. Representative hematoxylin and eosin stained sections for separation of dysplastic and non- dysplastic zone in mild/ moderate and severe dysplasia. i. Mild dysplasia; ii. Moderate dysplasia; iii. Severe dysplasia. Red line represents the line of application of laser beam for separating dysplastic and non- dysplastic zones. c. Representation agarose gel image showing methylation by Methylation Specific Restriction Analysis (MSRA). U: Undigested; H: HpaII digested. d. Representation agarose gel image showing methylation by Methylation Specific PCR (MSP). U: Unmethylated; M: Methylated. e. Histograms depicting percentage of methylation obtained for i. RBSP3; ii. LIMD1. f. Histograms depicting percentage of methylation in etiological groups in i. RBSP3; ii. LIMD1. p value (Fisher’s exact) represents the level of significance during univariate comparison. # Represents sample number; B: Basal; P: Parabasal: S: Spinous: T: Tumor; N: Normal. Group 1: HPV-TOB-; Group 2: HPV+TOB-; Group 3: HPV-TOB+; Group 3: HPV+TOB+.</p

Incidence of genetic deletion.

<p>Study of deletion of the genes in dysplasia and HNSCC. a. Representative autoradiograms and agarose gel showing the different types of alterations observed. Type of marker used is given below. LOH: Loss of heterozygosity; MA I: Microsatellite size alteration in one allele; MA II: Microsatellite size alterations in both alleles; LMA: Loss of heterozygosity in one allele and microsatellite size alteration in the other allele. # represents sample number. Arrowhead represents loss of allele; Star alteration change in allele size. b. Histogram representing alterations of the genes in dysplasia and HNSCC. i. RBSP3 ii. LIMD1 iii. CDC25A c. Percentage of co-alterations (deletion+ methylation) of the genes during progression of HNSCC. d. Co-deletion of LIMD1 and CDC25A. e. Histogram representing alterations of the genes in HNSCC in etiological groups I- IV. p value (Fisher’s exact) represents level of significance.</p

Experimental work flow and sample usage.

<p>Schematic diagram representing of type, distribution and usage of samples in different experimental procedures and etiological groups. <i>N</i>: Number of samples; PBL: Peripheral blood lymphocytes; HNSCC: Head and neck squamous cell carcinoma; IHC: Immunohistochemistry. HPV: Human papilloma virus; TOB: Tobacco. a. Normal specimens. b. Head and neck lesions.</p

Clinico-pathological features of pre-malignant (dysplasia) and invasive (squamous cell carcinoma) head and neck lesions.

<p>Abbreviations: n: Number of samples; BM: Buccal mucosa; ALV: Alveolus; TON: Tongue -ve: Factor absent; +ve: Factor present. p value represents level of significance during comparison.</p

Overall concordance obtained between different experiments undertaken in dysplastic lesions.

<p>Abbreviations: EXPR: Expression (IHC); METH: Methylation (promoter); DEL: Deletion; OVERALL: overall alterations (Deletion+ Methylation);DYS: Dysplasia; HNSCC: Head and neck squamous cell carcinoma; H: High; M: Moderate; L: Low; ND: Not done; NOR: Normal; TUM: Tumor; B: Basal; P: Parabasal; S: Spinous. p value represents level of significance.</p

Increased Heating Efficiency and Selective Thermal Ablation of Malignant Tissue with DNA-Encased Multiwalled Carbon Nanotubes

Nanoparticles, including multiwalled carbon nanotubes (MWNTs), strongly absorb near-infrared (nIR) radiation and efficiently convert absorbed energy to released heat which can be used for localized hyperthermia applications. We demonstrate for the first time that DNA-encasement increases heat emission following nIR irradiation of MWNTs, and DNA-encased MWNTs can be used to safely eradicate a tumor mass <i>in vivo</i>. Upon irradiation of DNA-encased MWNTs, heat is generated with a linear dependence on irradiation time and laser power. DNA-encasement resulted in a 3-fold reduction in the concentration of MWNTs required to impart a 10 °C temperature increase in bulk solution temperature. A single treatment consisting of intratumoral injection of MWNTs (100 μL of a 500 μg/mL solution) followed by laser irradiation at 1064 nm, 2.5 W/cm² completely eradicated PC3 xenograft tumors in 8/8 (100%) of nude mice. Tumors that received only MWNT injection or laser irradiation showed growth rates indistinguishable from nontreated control tumors. Nonmalignant tissues displayed no long-term damage from treatment. The results demonstrate that DNA-encased MWNTs are more efficient at converting nIR irradiation into heat compared to nonencased MWNTs and that DNA-encased MWNTs can be used safely and effectively for the selective thermal ablation of malignant tissue <i>in vivo</i>