HPV16 oncoproteins induce MMPs/RECK-TIMP-2 imbalance in primary keratinocytes: possible implications in cervical carcinogenesis

Abstract

Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome.

Figure 1. HPV-16 E7 up-regulates MMP-9 activity in organotypic cultures.

Primary HFKs were transduced with pLXSN-based retroviral vectors expressing HPV16 E6wt and/or E7wt. Cells were then differentiated in organotypic cultures for 9 to 11 days. A, Determination of gelatinase activity in organotypic cultures homogenates (epidermis separated from dermis). Equal amounts of proteins were loaded. Note the increase in MMP-9 gelatinase activity in HPV-16 E7wt and E6E7- expressing epidermis. B-B′, densitometry of pro-MMP-9 bands in epidermis and dermis, respectively. C, MMP-9 levels in epidermis homogenates were determined by Western blot. P<0.05.

Figure 2. HPV16 oncoproteins down-regulate RECK and TIMP-2 in HFKs.

A, Monolayer cultures of primary HFKs were transduced with pLXSN-based retroviral vectors expressing HPV16 E6wt and/or E7wt. RECK levels were determined by Western blot. Beta-actin was used as loading control. FF287 cell line was used as a positive control. B, Immunofluorescence detection of RECK (green) in HFKs expressing HPV oncoproteins. RECK is clearly detected in control and HPV16 E6wt expressing HFKs, while E7wt and E6E7-expressing cells exhibit reduced protein levels (arrows). Nuclei were counterstained with DAPI (blue). Original magnification 1000×. C, TIMP-2 was determined by ELISA (Biotrak). P<0.05.

Figure 3. Expression of HPV-16 E6/E7 down-regulates RECK in organotypic cultures.

A, Representative immunoreactivity of RECK in control and HPV16 E6E7 expressing rafts cultures. Note the strong RECK membrane staining pattern (black arrow) in control compared to the faint staining in HPV16 E6E7 expressing rafts cultures. B, Quantitative real-time PCR (qRT-PCR) of RECK expression in organotypic cultures. The relative expression levels were normalized to tubulin. Bars represent the means of triplicate experiments; bars, ±SE.

Figure 4. RECK is down-regulated in CIN II/III and invasive carcinoma samples.

Representative immunoreactivity of RECK in (A) Cervicitis, (B) cervical intraepithelial neoplasia II (CIN II), (C) cervical intraepithelial neoplasia III (CIN III) and (D) cervical invasive carcinoma. Note the strong RECK membrane staining pattern in cervicitis (black arrow), a diminished cytoplasmastic staining in CIN II/III and a very weak RECK staining in invasive carcinoma. Inset in (D): Sample incubated in the absence of primary antibody.