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HPV-16 impairs the subcellular distribution and levels of expression of protein phosphatase 1γ in cervical malignancy
- Takayuki Seiki1,
- Kazunori Nagasaka1Email author,
- Christian Kranjec2,
- Kei Kawana1,
- Daichi Maeda3,
- Hiroe Nakamura1,
- Ayumi Taguchi1,
- Yoko Matsumoto1,
- Takahide Arimoto1,
- Osamu Wada-Hiraike1,
- Katsutoshi Oda1,
- Shunsuke Nakagawa4,
- Tetsu Yano5,
- Masashi Fukayama3,
- Lawrence Banks2,
- Yutaka Osuga1 and
- Tomoyuki Fujii1
© Seiki et al.; licensee BioMed Central. 2015
Received: 31 May 2014
Accepted: 27 February 2015
Published: 7 April 2015
The high risk Human Papillomavirus (HPV) E6 oncoproteins play an essential role in the development of cervical malignancy. Important cellular targets of E6 include p53 and the PDZ domain containing substrates such as hScrib and Dlg. We recently showed that hScrib activity was mediated in part through recruitment of protein phosphatase 1γ (PP1γ).
Expression patterns of hScrib and PP1γ were assessed by immunohistochemistry of HPV-16 positive cervical intraepithelial neoplasm (CIN), classified as CIN1 (n = 4), CIN2 (n = 8), CIN3 (n = 8), cervical carcinoma tissues (n = 11), and HPV-negative cervical tissues (n = 8), as well as by subfractionation assay of the HPV-16 positive cervical cancer cell lines, CaSki and SiHa. To explore the effects of the HPV-16 oncoproteins, we have performed siRNA knockdown of E6/E7 expression, and monitored the effects on the expression patterns of hScrib and PP1γ.
We show that PP1γ levels in HPV-16 positive tumour cells are reduced in an E6/E7 dependent manner. Residual PP1γ in these cells is found mostly in the cytoplasm as opposed to the nucleus where it is predominantly found in normal cells. We have found a striking concordance with redistribution in the pattern of expression (9/11; 81.8%) and loss of PP1γ expression in HPV-16 positive cervical tumours (2/11; 18.2%). Furthermore, this loss of PP1γ expression and redistribution in the pattern of expression occurs progressively as the lesions develop (8/8; 100%).
Together, these results suggest that PP1γ may be a novel target of the HPV-16 oncoproteins and indicate that it might be a potential novel biomarker for HPV-16 induced malignancy.
Human Papillomaviruses (HPVs) are the aetiological agents of cervical cancer . This is caused by infection with the high risk subset of HPV types, of which HPV-16 is the most important, being responsible for over 60% of global cervical cancer cases . Cancer-causing HPVs encode two oncoproteins, E6 and E7, whose continued expression and activity is essential for maintaining the malignant phenotype, many years after the initial immortalising events [3,4]. Both viral oncoproteins function by perturbing the normal activity of a variety of different cellular control mechanisms. HPV E7 promotes cell cycle progression, in part through its association with members of the pocket protein family of tumour suppressors , whilst HPV E6 counteracts the pro-apoptotic effects of E7 through targeting the p53 tumour suppressor . In both cases, the viral oncoproteins make efficient use of the cellular ubiquitin-proteasome machinery, with E7 targeting pRb through the cullin 2 ubiquitin ligase complex , whilst E6 uses the E6AP ubiquitin ligase to target p53 . The effects of E6 and E7 are therefore cooperative, and this is reflected both in tissue culture systems, where they cooperate in the immortalisation of primary keratinocytes [9-11], and in animal models of tumourigenesis, where they cooperate in the induction of tumours in the skin and cervix [12,13].
Whilst targeting the pRb and p53 pathways is obviously very important for cervical tumourigenesis, it is also clear that E6 and E7 have a large number of other activities, many of which are also important for tumour development. In the case of high risk HPV E6 oncoproteins, an intriguing class of targets that appear to be important for HPV E6 induced malignancy are the PDZ (PSD/Dlg/ZO) domain containing substrates [14,15]. These are bound by E6 via a short stretch of amino acids within the extreme carboxy terminal region of the E6 oncoprotein. Most importantly, this PDZ binding motif (PBM) is only found in the high risk HPV E6 oncoproteins and is absent from the benign HPV E6 proteins [16,17]. Through this PBM, E6 can interact with a large number of cellular PDZ domain containing proteins, many of which are subject to E6-induced proteasomal degradation and E6-induced redistribution [16,18-21]. One of the most important of these targets is the cellular tumour suppressor hScrib. In Drosophila Scrib was originally identified as a potential tumour suppressor , and more recent studies in mammalian tissues also indicate tumour suppressive potential for hScrib. Loss of Scrib cooperates with c-Myc in the development of mammary carcinogenesis and Scrib also downregulates ERK signaling, with hScrib deregulation correlating with poor cancer prognosis [23-27]. In cervical tumourigenesis, hScrib patterns of expression are also perturbed as lesions develop, with hScrib being completely absent in many late stage tumours . We recently found that hScrib could interact with PP1γ  a protein phosphatase that plays a critical role in controlling chromatin organization and also has an important role in the DNA damage response pathway [30,31] This suggested that PP1γ expression patterns in cervical tumourigenesis might likewise be perturbed. Therefore we initiated a series of studies to investigate the pattern of PP1γ expression in HPV16 positive cervical tumours and derived cell lines. We show that PP1γ is indeed subject to a striking alteration in both its levels of expression and localisation, both as lesions develop, and in the tumour derived cell lines. However this altered pattern of expression is independent of hScrib, is due directly to E6/E7 expression, and highlights PP1γ as potential novel biomarker of HPV induced neoplasia.
Cell lines and culture
HPV positive cervical cancer cell lines, CaSki, SiHa and HeLa plus HPV negative C33A (cervical cancer derived) and HaCaT (human keratinocytes) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO2 . The effect of proteasome inhibitor was determined 24 hours post-transfection after 3 hours of treatment with 10 μM MG132 (Calbiochem).
For plasmid transfection, 293 cells were transfected using TransIT-293 transfection reagent (Mirus Bio) and HaCaT cells were transfected using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions, with pcDNA-HPV-16 E6. A plasmid expressing β-galactosidase was included in each transfection and pcDNA was used to equalize the input DNA.
The following commercial antibodies were used at the dilution indicated: anti-hScrib goat polyclonal antibody (Santa Cruz WB 1:1000, IHC 1:100), anti-PP1γ goat polyclonal antibody (Santa Cruz WB 1:1000), anti-PP1 Gamma/PPP1CC Antibody LS-B4960 IHC-plus (tm) rabbit polyclonal antibody (Lifespan bioscience, Inc. IHC 1:200), anti-PP1γ sheep polyclonal antibody (Abcam, WB 1:1000), anti-actin monoclonal antibody (Sigma, WB 1:5000), mouse monoclonal anti-p53 (DO-1) (Santa Cruz WB 1:500), anti-p84 mouse monoclonal antibody (Abcam, WB 1:1000), anti-E-Cadherin rabbit polyclonal antibody (Santa Cruz WB 1:500), anti-α-tubulin mouse monoclonal antibody (Abcam, WB 1:1000), mouse monoclonal anti-vimentin antibody (Santa Cruz WB 1:500).
The HPV-positive cervical cancer cells were seeded on 6 cm dishes and transfected using Lipofectamine 2000 (Invitrogen) with control siRNA against Luciferase (siLuc), or siRNA against HPV-16 and 18 E6 sequences (Dharmacon) described previously by Kranjec C et al., 2011. 72 hours post-transfection cells were harvested and total cell extracts or cell fractionated extracts were then analysed by western blotting. Alexa 568 labeled negative control siRNA (Qiagen) was used to measure transfection efficiency. The transfection efficiency was determined to be over 70% for each cell line.
Subcellular fractionation assays
Differential extraction of the cells to obtain cytoplasmic, membrane, cytoskeleton, and nuclear fractions was performed using the Calbiochem Proteo Extract Fractionation Kit according to the manufacturer’s instructions. To inhibit phosphatase activity during the preparation of cell lysates, phosphatase inhibitors (1 mM Na3VO4, 1 mM β-Glycerophosphate, 2.5 mM Sodium Pyrophosphate, 1 mM Sodium Fluoride) were also included.
Total cellular extracts were prepared by directly lysing cells from dishes in SDS lysis buffer. Alternatively cells were lysed in either E1A buffer (25 mM HEPES pH 7.0, 0.1% NP-40, 150 mM NaCl, plus protease inhibitor cocktail; Calbiochem) or RIPA buffer (50 mM Tris HCl pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, plus protease inhibitor cocktail; Calbiochem). For western blotting, 0.45 μm nitrocellulose membrane (Schleicher and Schuell) was used and membranes were blocked for 1 hour at 37°C in 10% milk/PBS followed by incubation with the appropriate primary antibody diluted in 10% milk/0.5% Tween 20 for 1 hour. After several washings with PBS 0.5% Tween 20, HRP-conjugated secondary antibodies (DAKO) in 10% milk/0.5% Tween 20 were incubated for 1 hour. Blots were developed using Amersham ECL reagents according to the manufacturer's instructions.
All tissue samples were fixed in formalin and embedded in paraffin (obtained from patients under Institutional Review Board approval through the University of Tokyo Hospital). For all antibodies, immunostaining was performed according to standard techniques using an autostainer (BenchMark XT; Ventana Medical Systems, Inc., Tucson, AZ, USA). Immunoreactivity was interpreted based on the negative control, which was incubated without the primary antibody. Detection of hScrib expression was evaluated based on the existence of basolateral membrane staining as described previously . For PP1γ, the expression was evaluated by nuclear staining. The immunostaining patterns of each sample were evaluated independently and blindly by pathologists specializing in gynaecological pathology, and cytology.
PCR-based HPV DNA testing
DNA was extracted from cervical smear samples by using the QIAGEN® DNeasy® Blood & Tissue Kits. PCR-based HPV DNA testing was performed using the PGMY-CHUV assay. Briefly, standard PCR was conducted using the PGMY09/11 L1 consensus primer sets and HLA-dQ primer sets. Reverse blotting hybridization was subsequently performed as described previously .
Distribution patterns of hScrib and PP1γ in HPV-16 positive cervical intraepithelial neoplasm (CIN) and cervical carcinoma tissues
Immunostaining patterns for hScrib and PP1γ in clinical samples of human uterine cervix
These results indicate that hScrib and PP1γ, whilst both being perturbed during the progression to malignancy, are altered in a manner that is not interdependent, suggesting that PP1γ might be an independent marker for cervical tumour development. Indeed, the pattern and expression levels of PP1γ declined with an almost linear relationship from normal tissue, through increasing grades of CIN lesion, to invasive cancer.
Analysis of PP1γ expression in HPV-16-positive cells
These results suggest that loss of nuclear PP1γ expression in HPV positive tumour cells is a direct result of the expression of the HPV E6/E7 oncoproteins.
PP1γ is subject to degradation in HPV-16 positive cells
PP1 is a major serine/threonine protein phosphatase, normally regulating the phosphorylation status of a large number of important cellular regulatory proteins [36-39]. Important activities include the regulation of chromosome structure during mitosis and also following DNA damage, through de-phosphorylation of histones [30,40], and the control of centrosome disjunction through antagonism of Nek2A kinase activity .
In this study we have identified PP1γ as a potential new biomarker of HPV-16 induced malignancy. Using HPV-16 positive cervical tumour derived cell lines, IHC analysis of HPV-16 positive cervical tumours and CIN lesions, we present compelling evidence that PP1γ expression patterns are perturbed as a result of infection with HPV-16.
We originally considered that the PP1γ/hScrib complex might be a general target for HPV-16 E6, based on our previous studies showing complex formation between hScrib and PP1γ. However, analysis of the expression patterns of PP1γ and hScrib in cervical tissues indicate that this is not the case. Most importantly however, this highlights PP1γ as an independent target of the HPV-16 oncoproteins. In the normal cervix, PP1γ is expressed throughout the differentiating cervical epithelium, with a predominantly nuclear pattern of expression, which is consistent with previous studies . To our surprise, we found that in all the HPV-16 positive cervical tumours analysed, this nuclear localisation of PP1γ was undetectable. Low levels of PP1γ can still be found within the cytoplasm of many cells within the majority of the cervical tumours that we analysed, although in 2/11 cases all PP1γ expression appeared to be lost. Similarly, perturbation in the pattern of PP1γ expression is apparent in CIN2 lesions, and this becomes more marked as the lesions progress to CIN3, suggesting that perturbation in the pattern of PP1γ expression is an early event in the development of cervical malignancy.
In order to understand whether these effects on PP1γ expression patterns were a direct consequence of E6/E7 activity, we then focused our attention on cells derived from HPV-16 positive cervical tumours. Again we found striking parallels with the IHC data, with very little PP1γ expression in the nucleus of HPV-16 positive CaSki or SiHa cells. In contrast, readily detectable nuclear PP1γ was observed in HaCaT cells. Most strikingly, siRNA ablation of E6/E7 expression resulted in a dramatic rescue of PP1γ expression within the nucleus of the HPV-16 positive cells, which appeared very similar to the effects seen upon the pattern of p53 expression. In contrast to p53 however, the alteration in the levels and pattern of PP1γ expression by E6 does not appear to involve the proteasome in cells derived from cervical tumours. Obviously further studies will be required to elucidate the precise mechanisms by which HPV-16 targets PP1γ.
Currently we have no information as to whether the HPV-16 E6/E7 oncoproteins can modulate any of these phosphorylation events in a PP1γ dependent manner, it is nonetheless intriguing that all of these pathways are perturbed to some extent in cells containing the HPV-16 oncoproteins. Future studies will investigate these aspects further, but it is tempting to speculate that targeting of the nuclear forms of PP1γ might contribute directly towards the generation of genome instability, chromatin remodeling and tumour progression. The cellular redistribution of PP1γ seems to have an important role in the development of centrosome abnormalities and chromosomal instability at early stages of cervical carcinogenesis. Taken together this study highlights the potential value of PP1γ as a novel biomarker for HPV-induced cervical neoplasia.
The authors are grateful to Kei Sakuma (Department of Pathology, Graduate School of Medicine, The University of Tokyo) for technical support on the preparation of IHC staining. Additionally, we thank Michihiro Tanikawa, Yuichiro Miyamoto, Kenbun Sone, Yuriko Uehara, Yuji Ikeda, Aki Miyasaka, Takahiro Koso, Tomoko Kashiyama, Tomohiko Fukuda, Kanako Inaba, Satoko Kojima, and Kensuke Tomio for their support and assistance. We also gratefully acknowledge the particular assistance of all members in Lawrence Banks's lab, and valuable comments on the manuscript from Miranda Thomas and David Pim. This work was supported by a Grant-in-Aid for Scientific Research (K.N.) from the Ministry of Education, Science and Culture, Japan, and in part by a research grant from the Associazione Italiana per la Ricerca sul Cancro (L.B).
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