Level of phospho-STAT3 (Tyr705) correlates with copy number and physical state of human papillomavirus 16 genome in cervical precancer and cancer lesions

Abstract

Our earlier studies indicated an important role of inducible transcription factor STAT3 in the establishment of persistent infection of human papillomavirus (HPV) type 16 and promotion of cervical carcinogenesis. Since HPV load and its physical state are two potential determinants of this virally-induced carcinogensis, though with some exceptions, we extended our study to examine the role of active STAT3 level in cervical precancer and cancer lesions and it's association with HPV viral load and physical state. An elevated level of active STAT3 was measured by assessing phospho-STAT3-Y705 (pSTAT3), in tumor tissues harboring higher viral load irrespective of the disease grade. Physical state analysis of HPV16 by assessing the degree of amplification of full length E2 and comparing it with E6 (E2:E6 ratio), which predominantly represent episomal form of HPV16, revealed low or undetectable pSTAT3. A strong pSTAT3 immunoreactivity was found in tissues those harbored either mixed or predominantly integrated form of viral genome. Cumulative analysis of pSTAT3 expression, viral load and physical state demonstrated a direct correlation between pSTAT3 expression, viral load and physical state of HPV. The study suggests that there exists a strong clinical correlation between level of active STAT3 expression and HPV genome copy number, and integrated state of the virus that may play a pivotal role in promotion/maintanence of tumorigenic phenotype.

Fig 1. Status of pSTAT3, along with the physical state and viral load of HPV16 in different cervical pre-cancer and cancer tissues.

Representative photograph showing levels of active pSTAT3(Y705) and total STAT3 in HPV16 positive cervical lesions. Cellular proteins (40μg/lane) derived from representative cervical tissues with indicated physical state of HPV16 genome (episomal- Epi; mixed–Mix; or integrated–Int) and viral load either lower (Low) or higher (High) than the median value of respective stage of disease were examined for pSTAT3 (Y705) and STAT3 expression by immunoblotting. Blots were stripped and re-probed with β-actin to equate the variations in cellular protein input.

Fig 2. Distribution of HPV16 viral load and pSTAT3 levels in cervical pre-cancer and cancer lesions.

Each circle represents individual LSIL, HSIL, or cancer tissues (SCC) with respective viral load and pSTAT3(Y705) level as determined by real-time PCR and immunoblotting. Viral load values are normalized per cell genome equivalent as described in Methods. ¹Arbitrary level of STAT3 expression in immunoblotting: Strong = (+++), Moderate = (++), Weak = (+), Nil/undetectable = (-). The vertical red line and horizontal blue line represent median values of STAT3 expression and viral load in tissues of each disease stage, respectively. GE, genome equivalents.

Fig 3. Analysis of the physical state of HPV16 genome and level of active pSTAT3(Y705) in cervical pre-cancer and cancer cases.

Distribution of normalized HPV16 E2: E6 ratio and level of pSTAT3 expression in cervical pre-cancer (LSIL and HSIL) and cancer cases (SCC). Each circle indicates an individual case. Normalized E2: E6 ratios were determined by PCR and calculated as described in Methods. Normalized E2: E6 ratio = 0 represents predominantly integrated HPV16 genome, value = 1 represents predominantly episomal viral genome, and values between 0 and 1 indicate a mixed form of HPV16 genome. ¹Arbitrary level of pSTAT3 expression in immunoblotting: Strong = (+++), Moderate = (++), Weak = (+), Nil/undetectable = (-). The vertical red line and horizontal blue line represent median values of pSTAT3 expression and E2: E6 ratio in each disease group, respectively.