KSHV co-infection regulates HPV16+ cervical cancer cells pathogenesis in vitro and in vivo

Abstract

High-risk human papillomavirus (HPV) infection is the etiological agent of cervical, oral and oropharyngeal cancers. Another oncogenic virus, Kaposi sarcoma-associated herpesvirus (KSHV) can cause several human cancers arising in those immunocompromised patients. KSHV DNA has been detected in the oral cavity and the female genital tract, although its detection rate in cervical samples is relatively low. Therefore, it remains unclear about the role of KSHV co-infection in the development of HPV-related neoplasia. We recently report that KSHV infection of HPV16+ cervical cancer cell line SiHa induces several pro-inflammatory factors production while reducing HPV16 E6 and E7 expression through the manipulation of cellular microRNA function. In the current study, we focus on determining the influence of KSHV co-infection on cervical cancer cells pathogenesis in vitro and in vivo. We found that KSHV co-infection is able to maintain SiHa and/or CaSki cells pathogenesis and tumorigenesis, although hijacking HPV oncogenic proteins expression. In mechanisms, KSHV co-infection is capable of increasing Macrophage migration inhibitory factor (MIF) and its receptor CXCR2 expression from cervical cancer cells, which may contribute to cervical cancer development. Our data indicate that KSHV co-infection may act as a potential co-factor to promote HPV-related neoplasia development.

Keywords: HPV; KSHV; MIF; SiHa.

Figure 1

KSHV co-infection does not affect SiHa cell growth and viability. A. SiHa were incubated with purified KSHV (MOI~10), or medium control (mock) for 2 h. After cells were incubated for indicated additional time, cell proliferation was measured using the WST-1 assays. B, C. Cell viability was measured by using flow cytometry as described in the Methods. Error bars represent the S.D. for 3 independent experiments.

Figure 2

Comparison of cell invasiveness and anchorage-independent growth abilities between mock and KSHV co-infected SiHa cells. A. SiHa were incubated with or without purified KSHV (MOI~10) for 2 h, after additional 48 h incubation, the transwell assays were performed to determine cell invasiveness ability as described in the Methods. B. The anchorage-independent growth ability was determined using the soft agar assays as described in the Methods.

Figure 3

Comparison of tumorigenesis ability between mock and KSHV co-infected SiHa cells in a nude mice xenograft model. A, B. The mock and KSHV co-infected SiHa cells (approximately 5 × 10⁵ cells were mixed at a ratio of 1:1 with growth factor-depleted Matrigel) were injected subcutaneously into the right flanks of nude mice, respectively. The mice were observed and measured every 2~3 d for the presence of palpable tumors for ~40 d. Error bars represent the S.D. from 2 independent experiments. C. Protein expression within tumor tissues from representative injected mice was measured by using immunohistochemistry staining as described in the Methods.

Figure 4

The up-regulation of MIF and its receptor by KSHV co-infection in vitro and in vivo. A. SiHa were incubated with purified KSHV (MOI~2 or 10), or medium control (mock) for 2 h. After cells were incubated for additional 72 h, MIF concentrations in supernatant were determined by using ELISA. Error bars represent the S.D. for 3 independent experiments, **=P < 0.01. B. The mock and KSHV co-infected SiHa cells were injected subcutaneously into the nude mice as described previously. Protein expression within tumor tissues from representative injected mice was measured by using immunohistochemistry staining.