Presence Of Beta Human Papillomaviruses In Nonmelanoma Skin Cancer From Immunocompetent Patientsand Its Mechanism

Non-melanoma skin cancer (NMSC) are the most common types of cancer includingSquamous cell carcinoma (SCC), basal cell carcinoma (BCC), actinic keratosis(AK),keratoacanthoma mesothelioma and so on. SCC accounts for the most of NMSC. In recentyears, the incidence of NMSC was on a rapid and worrisome increase in China, whichsignificantly impairs the health and life quality. And the resulting social burden and healthcare burden can not be ignored. The reported factors of NMSC include older age, skincolor, cutaneous human papillomavirus (HPV) infection, exposure to UV radiation,immunosuppression (such as organ transplant recipients, patients with AIDS) and so on.Previous studies have demonstrated that HPV infection, UV-induced damage are the two major risk factors for NMSC. HPVs are small, circular, epitheliotropic, double-strandedDNA viruses. More than130subtypes of HPV have been sequenced so far. HPVs aresubdivided into16different generas such as alpha, beta and gamma HPV. α-HPV has beenverified to contribute to cervical cancer, while β-HPV are exclusively cutaneous types, asubset of which probably plays a important role in the development of NMSC. However,association between of β-HPV and NMSC remains unclear.Unlike α-HPV, studies of pathogenic mechanism of β-HPV have just started.Previous studies have shown β-HPV and UV jointly contribute to NMSC. Tumorigenesisis associated with a variety of factors in which viral infections is one of the importantbiological factors, but the mechanism is unkonwn. Autophagy is widely present ineukaryotic cells, belonging to a type II programmed cell death. Autophagy is an importantdefensive and protective mechanisms through phagocytose and degrade damaged cells andpathogenic microorganisms as a―scavenger‖. Currently, little is known about whetherβ-HPV modulates autophagy. Autophagy was reported to play dual roles in tumorprogression. In the early stage of tumor, autophagy is a tumor suppressive mechenism. Infuture research, it is most important to actively use autophagy to inhibit cancer cellproliferation and to maximize curb its cancer-promoting effect.To analyse the presence of β-HPV in northwestern China, We collected192patientswith NMSC and80healthy subject to detect β-HPV DNA. Further more, we use specificPCR for β-HPV genotyping to clarify potentially pathogenic high-risk subtypes. Then, weuse immunohistochemical to detect expression of autophagy and related signalingpathways in order to explore the pathogenesis of autophagy in NMSC.Objectives:1. To assess the presence of β-HPV in NMSC from Northwestern China.2. To clarify the high-risk subtypes of β-HPV in the development of NMSC.3. To explore pathogenic mechanism of autophagy induced by β-HPV infection inNMSC.Methods: First of all, we isolated β-HPV DNA from192NMSC and80healthy subject bynested PCR. We also examined the α-HPV DNA in the samples by PCR. β-HPV wasdetected from paraffin tissue by the same nested PCR to compare for biopsies. Secondly,typing for12kinds of β-HPV subtypes was perfomed on β-HPV positive patients. Afterdetermined β-HPV subtypes, we investigated the autophagy level and the expression ofrelated signaling pathways’s markers through immunohistochemical techniques. Finally,we use transmission electron microscopy (TEM) to verify autophagy level changes inNMSC.Results:1. Analysis of β-HPV epidemic in NMSC in Northwestern China: Nested PCR for skintissue β-HPV DNA detection showed that the positive rate of72SK,50AK,20SCC,50BCC and80normal skin respectively are21%、80%、75%、44%、35%. And, positive rateof AK and SCC is much higher than the normal skin (P <0.05), ORAK=8.75, ORSCC=5.469.Compared with normal skin, in SK and BCC the detection rate was not statisticallysignificant.2. α-HPV presence in the different lesions: In α-HPV detection, there was no significantdifference between cases and control groups;1patient in AK,1in SCC and2lesions innormal skin were multiple genotype infection positive. No mixed infections wereobserved in SK and BCC.3. β-HPV DNA detection in paraffin tissue: Nested PCR displayed that β-HPV DNAwas detected in15(62.5%) of24AK,11(44%) of25SCC, and1(8.3%) of12normalskin. Despite the results have a significant difference compared with normal skin, but theβ-HPV positive rate is lower than the fresh tissue.4. β-HPV genotyping: We use12kinds of β-HPV (HPV5,8,15,17,19,20,21,23,36,38,49,80) specific PCR primers for positive samples were further typing. The resultsshowed that the most frequently found genotypes in AK(12/15,80%) and SCC(25/40,62.5%) was HPV38. However, the detection rate of HPV was lower in SK and normalskin. 5. Immunohitochemical detection of autophagy-related markers in NMSC:Immunohistochemical results showed that compared with normal controls, after HPVinfected (both HPV38+and HPV38-), the expression of Beclin1, LC3were significantlyhigher (P <0.05) than normal skin. Moreover, the level was highest in HPV38+SCC. Thelevel of p62were lower in controls (P <0.05).6. Immunohistochemical analysis of the PKR signaling pathway: Compared with thecontrol group, after HPV infected (both HPV38+and HPV38-), the expression of PKR,p-PKR, eIf2α, p-eIf2α were higher (P <0.05) than normal skin. Moreover, the level washighest in HPV38+AK.6. TEM observation of autophagosome in NMSC: The number of autophagosome inSCC were more than AK and normal control group (P <0.001).Major conclusions:1. β-HPV DNA is more prevalent in AK and SCC, which indicates that β-HPVinfection is closely correlated with the pathogenesis of AK and SCC.2. HPV38is the most frequently genotypes in AK and SCC, which indicates thatHPV38may be the high-risk type of β-HPV can contribute to NMSC.3. Autophagy was upregulaed in AK and SCC: It is suggesting that in the early stage (iethe precancerous lesions AK) of SCC as the level of autophagy increased, it can clarify thedamaged cells to maintain genomic stability play a tumor suppressor role. And in thedevelopment of SCC, excessive autophagy can provide nutrition and energy for celltransformation, and finally promote tumor surviving and proliferation in the harshenvironment.4. HPV38may increase autophagy level by activting the PKR-eIf2α signaling pathway.