Genomic profiling reveals the pivotal role of hrHPV driving copy number and gene expression alterations, including mRNA downregulation of TP53 and RB1 in penile cancer

Abstract

The incidence of penile cancer (PeCa) is increasing worldwide, however, the highest rates are reported in underdeveloped countries. The molecular mechanisms that underly the onset and progression of these tumors are still unclear. Therefore, our goal was to determine the genome-wide copy number alterations and the involvement of human papiloma virus (HPV) (TP53 and RB1), inflammatory (COX2 and EGFR), and PI3K/AKT pathway (AKT1, AKT2, EGFR, ERBB3, ERBB4, PIK3CA, and PTEN) associated genes in patients with PeCa from a high incidence region in Brazil (Maranhão). HPV genotyping was performed by nest-PCR and genome sequencing, copy number alterations (CNAs) by array comparative genomic hybridization and gene copy number status, gene, and protein expression by quantitative polymerase chain reaction, reverse transcriptasequantitative polymerase chain reaction, and immunohistochemistry, respectively.HPV genotyping revealed one of the highest frequencies of HPV reported in PeCa, affecting 96.4% of the cases. The most common CNAs observed were located at the HPV integration sites, such as 2p12-p11.2 and 14q32.33, where Abbreviations: aCGH, array comparative genomic hybridization; CNA, copy number alteration; Cq, quantification cycle; FFPE, formalin-fixed paraffin-embedded; HPV, human papiloma virus;HrHPV, High risk HPV; IHCi, mmunohistochemistry; PCR, polymerase chain reaction; PeCa, penile cancer; qPCR, quantitative polymerase chain reaction; RT-qPCR, reverse transcriptase-quantitative polymerase chain reaction; TMA, tissue microarray. ADAM 6, KIAA0125, LINC00226, LINC00221, and miR7641-2, are mapped. Increased copy number of ERBB3 and EGFR genes were observed in association with COX2 and EGFR overexpression, reinforcing the role of the inflammatory pathway in PeCa, and suggesting anti-EGFR and anti-COX2 inhibitors as promising therapies for PeCa. Additionally, TP53 and RB1 messenger RNA downregulation was observed, suggesting the occurrence of other mechanisms for repression of these oncoproteins, in addition to the canonical HPV/TP53/RB1 signaling pathway. Our data reinforce the role of epigenetic events in abnormal gene expression in HPV-associated carcinomas and suggest the pivotal role of HPV driving CNAs and controlling gene expression in PeCa. K E Y W O R D Scarcinoma of the penis, genomic alterations, human papillomavirus, molecular markers

Genome-Wide Profiling Reveals HPV Integration Pattern and Activated Carcinogenic Pathways in Penile Squamous Cell Carcinoma

“…In PSCC, Busso-Lopes et al [ 10 ] found that copy number variations (CNVs) of 19 genomic regions are commonly detected in HPV + tumors, including in chromosomal regions 2q33.2-q33.3 and 2q35. Macedo et al [ 13 ] also found several loci, such as 2p12-p11.2, 14q32.33, and 2p16.3, presented CNV in most of HPV+ PSCCs, and several CNVs were correlated with clinicopathological factors. However, none of these results could directly indicate whether these differences between HPV-positive and HPV-negative PSCCs were induced by virus.…”

“…The integration pattern and characteristics of HPV in the PSCC genome are also unclear. Several studies have indicated that HPV-positive PSCC harbors a different genomic profile [ 10 , 11 , 12 , 13 ] and specific epigenetic alterations [ 12 ] when compared to HPV-negative PSCC or normal tissue. For instance, HPV-positive PSCC often harbors an APOBEC mutation [ 11 ]; copy number variations (CNVs) of 19 genomic regions are commonly detected in HPV-positive tumors [ 10 ]; HPV-positive tumors are characterized by the widespread loss of DNA methylation [ 12 ].…”

Downregulation of miR-145 is associated with perineural invasion in penile carcinoma

“…Studies have demonstrated that EGFR is often overexpressed its protein and gene dysregulation, has already been associated with advanced stage, lower overall survival and metastatic lymph node status (34). Downregulation of miR-145 may be one of the mechanisms for high expression of EGFR, especially in HPV positive tumors (35). Additionally, this microRNA can regulate pathways such as Wnt, TGF-beta, Calcium which are involved in cell growth, progression, survival, angiogenesis, and apoptosis (23).…”

Systemic treatment of penile squamous cell carcinoma—hurdles and hopes of preclinical models and clinical regimens: a narrative review

“…Recently, Macedo et al performed array-CGH in 20 of 55 PeSCC (81.8% of them were HPV positive) (19). D e s p i t e o f n o t h a v i n g a n y s i g n i f i c a n t c l i n i c a lhistopathological association, a noteworthy number of altered chromosomal regions coincided with sites of HPV integration into the human genome.…”