PIK3CA-mediated PI3-kinase signalling is essential for HPV-induced transformation in vitro
© Henken et al; licensee BioMed Central Ltd. 2011
Received: 5 November 2010
Accepted: 10 June 2011
Published: 10 June 2011
High-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development. The additional (epi)genetic alterations driving malignant transformation of hrHPV-infected cells however, are not yet fully elucidated. In this study we experimentally assessed the role of the PI3-kinase pathway and its regulator PIK3CA, which is frequently altered in cervical cancer, in HPV-induced transformation.
Cervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal in vitro model system of hrHPV-transfected keratinocytes, representing the immortal and anchorage independent phenotype, was assayed for PI3-kinase activation and function using chemical pathway inhibition i.e. LY294002 treatment, and PIK3CA RNA interference. Phenotypes examined included cellular viability, migration, anchorage independent growth and differentiation. mRNA expression of hTERT and HPV16 E6E7 were studied using quantitative RT-PCR and Northern blotting.
Cervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPV-induced transformation in vitro, expression of the catalytic subunit PIK3CA as well as activation of downstream effector PKB/AKT progressively increased in parallel. Inhibition of PI3-kinase signalling in HPV16-transfected keratinocytes by chemical interference or siRNA-mediated silencing of PIK3CA resulted in a decreased phosphorylation of PKB/AKT. Moreover, blockage of PI3-kinase resulted in reduced cellular viability, migration, and anchorage independent growth. These properties were accompanied with a downregulation of HPV16E7 and hTERT mRNA expression. In organotypic raft cultures of HPV16- and HPV18-immortalized cells, phosphorylated PKB/AKT was primarily seen in differentiated cells staining positive for cytokeratin 10 (CK10). Upon PI3-kinase signalling inhibition, there was a severe impairment in epithelial tissue development as well as a dramatic reduction in p-PKB/AKT and CK10.
The present data indicate that activation of the PI3-kinase/PKB/AKT pathway through PIK3CA regulates various transformed phenotypes as well as growth and differentiation of HPV-immortalized cells and may therefore play a pivotal role in HPV-induced carcinogenesis.
It has been well established that high-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development . While the two most potent viral oncogenes E6 and E7 are necessary for the initiation and maintenance of cellular proliferation [2, 3], their expression is not sufficient for full transformation of epithelial cells. Hence, there are extensive efforts towards identifying additionally required host cell events. Chromosomal analysis has revealed that a gain of chromosome 3q is the most common event in the development of cervical squamous cell carcinoma (SCC) [4–6]. In fact, a gain of chromosome 3q was found in all SCC previously analysed by microarray CGH . Candidate oncogenes on chromosome 3q include the gene encoding p110α, the active subunit phosphatidylinositol 3-kinase catalytic alpha (PIK3CA) of class I PI3-kinase. Upon activation, PI3-kinase initiates events leading to phosphorylation of PKB/AKT, which affects additional downstream signalling proteins involved in survival and cell growth. Indeed, deregulation of the PI3-kinase pathway is common in many human malignancies . In cervical carcinomas, an increased copy number of PIK3CA was positively correlated with an increase in phosphorylated PKB/AKT, one of the downstream effectors . Additionally, the level of p-PKB/AKT expression increased proportional to the histopathological grade of (pre)malignant cervical diseases [9, 10]. Although it has been found that HPV16E7 can activate PKB/AKT in differentiating cells , the relevance of PI3-kinase signalling in the process of cervical cancer development following a transforming hrHPV infection remains to be experimentally explored. Moreover, no functional studies on the specific role of PIK3CA in cervical carcinogenesis have yet been performed.
Previously we have shown that in vitro transformation of primary keratinocytes mediated by full-length hrHPV was accompanied with a gain of chromosome 3q in immortalized descendants . This model system of HPV-transformed keratinocytes therefore provides interesting and useful source material to study the potential functional role of PI3-kinase for the various transformed phenotypes. In the present study we analysed PIK3CA expression in cervical squamous cell carcinomas. We also performed functional analyses of the contribution of PI3-kinase signalling, and specifically PIK3CA, to hrHPV-mediated transformation in vitro.
Cell culture, LY294002 treatment and transfection
Primary human foreskin keratinocytes, HPV16 and HPV18-immortalized keratinocyte cell lines (FK16A and FK18A) as well as HPV16E6E7 containing keratinocytes were cultured as described previously . The latter cells were generated by transduction of primary human foreskin keratinocytes with the retroviral vector pLZRSneo containing HPV16E6E7, as described previously .
FK16A cells between passages 45 and 62 represented immortal and anchorage dependent cells and FK16A cells between passages 99 and 189 represented anchorage independent cells . Prior to LY294002 (10 μM and 20 μM) (Cell Signaling Technology, Beverly, USA) or DMSO treatment, cells were starved overnight to ensure similar phosphorylation status. A pool of 4 siRNA sequences targeting PIK3CA (cat#L-003018-00-0005, Dharmacon, Lafayette, USA) was transfected using Dharmafect reagent 2 (Dharmacon) according to the manufacturers protocol. Pools of 4 non-targeting siRNAs (cat#D-001810-10-05) and PLK1 specific siRNAs (cat#L-003290-00-0005) were used as negative and positive controls, respectively. The use of a non-targeting siRNA pool ensures control for off-target effects. Transfection of cDNA encoding for myristoylated PIK3CA  Addgene, Cambridge, USA) and cotransfections with HPV16-URR luciferase constructs into FK16A cells were performed using Effectene (Qiagen, Hilden, Germany) according to instructions. Firefly luciferase and Renilla luciferase were measured using Dual Luciferase assay (Promega, Wisconsin, USA).
All tissue specimens were collected during the course of routine clinical practice at the Department of Obstetrics and Gynecology at the VU University medical center. Normal epithelial control samples were obtained from histologically normal frozen biopsies of non-cancer patients undergoing hysterectomy. This study followed the ethical guidelines of the Institutional Review Board of the VU University medical center.
RNA isolation, RT-PCR and Northern Blotting
Isolation of mRNA from cell lines was performed using RNA-B reagent (Tel-Test, Friendswood, USA) and DNase treated (Promega) prior to cDNA synthesis using specific reverse primers (see below). Total RNA from micro-dissected frozen biopsies of cervical SCCs and normal ectocervical controls was isolated using Trizol reagent (Invitrogen Life Technologies, Breda, The Netherlands) as described before . Quantitative RT-PCR was performed as described previously  using the following primers for PIK3CA forward 5'-CCTGATCTTCCTCGTGCTGCTC-3' and reverse 5'- ATGCCAATGGACAGTGTTCCTCTT -3' using SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA). And for hTERT forward 5'-CACGCGAAAACCTTCCTCA -3', reverse 5'-CAAGTTCACCACGCAGCC-3' and the probe FAM-5'-CTCAGGGACACCTCGGACCAGGGT -3'-TAMRA using Universal PCR Master Mix (Applied Biosystems). To correct for RNA quality and input, we performed RT-PCR for the housekeeping gene snRNP as described before in cell line experiments . For quantification, a standard curve was established using serial dilutions of cervical cancer cell line cDNA. To determine HPV16E7 mRNA expression LightCycler real-time PCR assays were applied as described before [17, 18] as well as Northern Blotting for HPV16. Total RNA was separated on a 1% agarose gel, blotted on nylon membranes (GeneScreen, PerkinElmer Life Sciences, Waltham, USA) and hybridized with a radioactive labelled full length HPV16 probe.
Antibodies against total (cat#9272) or phosphorylated forms of PKB/AKT (cat#4058), PIK3CA (cat#4255) and loading control beta-actin (cat#4967) (all 1:1000 from Cell Signaling Technology) were used according to the manufacturers instructions. Membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies and the levels of corresponding proteins were visualized using SuperSignal West Dura Extended Duration Substrate (Pierce).
Immunohistochemical staining and immunofluorescence assays
Immunohistochemical staining was performed using 4 μm sections which were deparaffinised, rehydrated and microwave-treated (800W) for 10 min in Tris buffer (pH9), followed by incubation for 30 min in 3% H2O2 in methanol. Antibody incubation with PIK3CA (cat#4249 Cell Signaling 1:200) was performed overnight at 4°C and for detection the EnVision horseradish peroxidase system (Dako, Heverlee, Belgium) was used.
For immunofluorescence, 4 μm sections were rehydrated, treated with 10 mM citrate, pH 6.0 at 95°C for 10 min and allowed to cool to room temperature over 20 min. Slides were then treated with 3% H2O2 in water and blocked in 1XPBS containing 10% goat serum. P-PKB/AKT and Ki-67 were detected by sequential probing with respective antibodies because both were raised in rabbit. P-PKB/AKT was probed with rabbit monoclonal antibody (cat#2118-1, Epitomics Inc, 1:100 dilution) and detected by fluorescine-conjugated tyramide (NEL701001, PerkinElmer Life Science, USA) as per the manufacturers direction. Subsequently, Ki-67 was probed with rabbit monoclonal antibody (ab16667, Abcam, 1:100 dilution) and detected by Alexa Fluor 555 conjugated anti-rabbit IgG (cat#A21429, Invitrogen-Molecular Probes, USA.) as per the manufacturers protocol. P-PKB/AKT and CK10 localization was detected by concurrent probing with respective primary antibodies as they were raised in different species. Raft sections were treated with rabbit anti p-PKB/AKT (described above) and mouse monoclonal anti-CK10 (cat#ab1421, 1:150 dilution, Abcam, Cambridge, USA). Anti-CK10 was detected with Alexa Fluor 555 conjugated goat anti-mouse IgG (cat#A21424, Invitrogen-Molecular Probes, USA). Finally slides were mounted with DAPI containing media (VECTASHIELD, H1200, Vector Laboratories, USA), viewed under Olympus AX70 microscope fitted with Chroma filters. Photomicrographs were captured by Axiovision camera at 20× magnifications of objective and finally processed with Photoshop CS2 (Adobe) for documentation.
Cell Viability, Migration, Anchorage Independent Growth
Cell viability was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye reduction (ICN Biomedicals Inc, USA). Cells were seeded in triplicate wells in 96-wells plates, transfected or starved overnight followed by LY294002 treatment and grown for 5 days. Control conditions were set to 100%.
For cellular migration assays cells were plated at high confluence and uniformly scratched to create a cell-free gap. After 24-48 hours in serum-free keratinocyte growth medium, plates were examined and photographed to asses the migration of neighbouring cells into the gap.
To examine anchorage independent growth under the different conditions 5000 cells of each condition were plated in semi-solid agarose (as described previously ). After 3 weeks colonies larger than ~50 cells were counted and pictures taken.
Organotypic raft cultures
The HPV containing keratinocytes were grown as epithelial raft tissues as described previously . For all conditions duplicate rafts were developed. Transfections with siRNAs were carried out the day before seeding on the collagen beds. Inhibitor treatment was started after seeding and continued throughout the 9 days of culturing at the liquid-air interface. After harvesting, the raft tissues were fixed in formalin and embedded in paraffin. For histological examination, 4 μm sections were stained with hematoxylin and eosin.
All statistical analyses were carried out using the T-test in the SPSS software package (SPSS 15.0, Chicago, USA).
Increased PIK3CA expression in cervical carcinomas
The PI3-kinase pathway becomes increasingly activated during hrHPV-induced transformation in vitro
To examine whether the PI3-kinase pathway is relevant for HPV-mediated transformation in vitro, we made use of our model system consisting of primary keratinocytes transfected with full-length HPV16. This longitudinal model consists of successive stages of transformation, including immortalization and anchorage-independent growth. The immortal cells have bypassed the senescence and crisis barriers that mark the end of the replicative and extended lifespan, respectively, and display increased telomerase activity . At this stage, the cells also possess a gain of chromosome 3q . Upon further passaging the immortal cells acquired an anchorage-independent phenotype .
Phosphorylation and activation of PKB/AKT is a result of upstream signalling events. Hence, we proceeded to determine whether we could influence PKB/AKT phosphorylation by modulating PI3-kinase activity in our model system. Different passages of FK16A cells were treated with the chemical PI3-kinase inhibitor LY294002, which fits in the ATP binding pocket of the catalytic subunit PIK3CA . LY294002 treatment resulted in reduction of the levels of phosphorylated PKB/AKT at all passages, while total PKB/AKT remained the same (Figure 2B). These results indicate that phosphorylation of PKB/AKT in these cells relies at least in part on the activity of PI3-kinase signalling.
PI3-kinase signalling is functionally involved in hrHPV-induced transformation in vitro
The catalytic subunit PIK3CA is essential for HPV-mediated transformation
Following PIK3CA specific siRNA transfection cells were seeded for the functional tests described above. Similar to LY294002 treatment, siRNA-mediated PIK3CA knockdown significantly reduced the number of viable FK16A cells (p = 0.0005) compared to transfection with a pool of non-targeting siRNAs (Figure 4B). Knock-down of PLK1, inducing cell death and used as a positive control, also resulted in a significant reduction of viable cells (p = 0.0003). FK16A cells transfected with siRNAs targeting PIK3CA also lost their migratory capacity and were unable to close the wound (Figure 4C). Primary human foreskin keratinocytes, included as controls, displayed a minor reduction in migratory capacity upon PIK3CA silencing (Additional File 3, Figure S3), in concordance with the lower level of PIK3CA mRNA in these cells (Figure 2A). Lastly, anchorage-independent growth was also affected by PIK3CA knockdown to a similar extent as after treatment with 10 μM LY294002 (p = 0.05) (Figure 4D). Taken together, these results show that PIK3CA is relevant for the oncogenic properties of HPV-mediated transformation.
PI3-kinase signalling is involved in transcriptional regulation of HPV
PI3-kinase signalling is essential for growth and differentiation of HPV-immortalized cells in organotypic cultures
Culturing human keratinocytes on organotypic raft mimics epithelial stratification and differentiation into various strata, whereby PI3-kinase signalling is known to play a role [10, 23–25]. We have previously shown that differentiation is disturbed in HPV16- and HPV18- immortalized keratinocytes, revealing phenotypes reminiscent of HPV-induced dysplastic lesions . Thus, we next studied the roles of PI3-kinase signalling in growth and differentiation of these HPV-immortalized keratinocytes in organotypic raft cultures. FK16A cells displayed a severely dysplastic morphology on epithelial raft cultures, whereas an HPV18 immortalized cell line, FK18A, appeared less dysplastic and maintained terminal differentiation . These different attributes permit a comparison of cell growth and differentiation upon modulating PI3-kinase. As described above, FK18A cells also exhibited increased levels of phosphorylated PKB/AKT with passaging (Additional File 1, Figure S1).
To further assess the relation between phosphorylated PKB/AKT, cell proliferation and squamous differentiation, we performed double indirect immunostainings for p-PKB/AKT and Ki-67, a proliferation marker, or cytokeratin 10 (CK10), a differentiation marker.
As shown in Figure 7B, in raft cultures of FK16A cells, the p-PKB/AKT signals were primarily detected in the cytoplasm, while Ki-67 was restricted to the nucleus. Both were detected stochastically throughout most of the epithelium. In the FK18A raft cultures, while Ki-67-positive cells were found in all cell strata, the p-PBK/AKT signal was primarily detected in the upper most differentiated strata. Interestingly, the two signals rarely co-localized to the same cells.
After chemical inhibition of PI3-kinase, a fraction of the remaining epithelial cells were positive for Ki-67 despite the fact that phosphorylated PKB/AKT was strongly reduced or nearly abolished. In the presence of siRNAs targeting PIK3CA, the p-PKB/AKT signals in FK16A raft cultures were also greatly reduced while Ki-67 signals were not altered. The non-targeting siRNAs had no effect on either. Upon ectopic expression of activated PIK3CA, more cells in the upper strata of the FK18A raft cultures were positive for p-PKB/AKT while the signals of Ki-67 appeared unchanged.
Figure 7C shows that, in both cell lines, differentiation marker CK10 was positive from the suprabasal layers upwards. Consistent with the high dysplastic histology (Figure 7A), CK10 expression was patchy in the FK16A raft cultures with packets of CK10-negative cells, whereas the signals were much more uniformly detected across the FK18A cultures (Figure 7C). After chemical inhibition of PI3-kinase signalling in FK16A and FK18A cultures, not only p-PBK/AKT was largely abolished, but also the fraction of CK10-positve cells and signal strengths were both greatly reduced, especially in FK16A cultures. Similarly, in the siPIK3CA-treated FK16A cultures, p-PKB/AKT was virtually abrogated while CK10 signals were greatly reduced. In contrast, the non-targeting siRNAs had no effect on either signal. These results strongly suggest that differentiation was largely abolished upon reduction of p-PKB/AKT. Despite an increased phophorylated PKB/AKT staining in FK18A cells overexpressing PIK3CA, CK10 staining patterns were similar to the non-transfected controls, because these cells were already strongly positive for CK10. These data confirm that PI3-kinase signalling is important for regulating differentiation of HPV-immortalized cells.
The PI3-kinase/PKB/AKT signalling pathway affects a wide variety of cellular characteristics such as proliferation, differentiation and cell survival and is often altered in many human malignancies . In cervical cancer, a gain of the long arm of chromosome 3, where PIK3CA is located, is often described and suggested to be a compulsory second hit for malignant transformation following an hrHPV infection [4, 6]. However, data is scarce on the biological effects of altered PIK3CA expression and PI3-kinase signalling in cervical carcinogenesis.
In the present study we showed that mRNA expression of the catalytic subunit PIK3CA was significantly upregulated in cervical SCC. In our previous studies using arrayCGH, up to 100% of cervical SCC were shown to contain additional copies of chromosome 3q . Additionally, low levels PIK3CA amplifications were found in 40%-74% of cervical carcinomas [5, 8, 26]. Bertelsen et al showed that an increase in PIK3CA copy numbers (more than 3 copies) was significantly correlated to elevated p-PKB/AKT expression in cervical SCC and high-grade precursor lesions . Also, other studies reported an increase in p-PKB/AKT staining with increase of histopathological grade of cervical disease [9, 10].
Functionally, we showed that PIK3CA and concomitant PI3-kinase signalling is involved in the different stages of HPV-mediated transformation in vitro. Modulating PI3-kinase activity in HPV transfected keratinocytes affected proliferation, migration, anchorage independent growth, and epithelial growth and differentiation in organotypic cultures. These data are consistent with previous reports using different cellular systems. For instance, in ovarian cancer cell lines PI3-kinase pathway inhibition with LY294002 resulted in reduced proliferation via cell cycle arrest in G1  or induction of apoptosis . Proliferation measured by cell number and [3H]-Thymidine incorporation was also reduced in rat intestinal epithelial cells as a result of LY294002 treatment . Furthermore, the involvement of PI3-kinase in colony formation has been shown in mammary epithelial cells where overexpression of PIK3CA increased colony formation while a dominant negative form of PIK3R1 (p85) lacking the PIK3CA binding domain repressed colony formation . This latter study also showed that, although PKB/AKT is the main effector of PI3-kinase, PKB/AKT activation cannot substitute PI3-kinase signalling.
Functional studies in cervical cells are restricted to the common hrHPV positive cervical cancer cell lines such as SiHa, HeLa and CaSki and did not include the explicit analysis of PIK3CA as a candidate oncogene. Treatment of SiHa with the PI3-kinase inhibitor LY294002 led to reduced proliferation and increased apoptotic DNA fragments . For HeLa and CaSki the reduction in proliferation upon LY294002 treatment was shown to be independent of apoptosis, though did sensitize the cells to radiation .
None of the previous studies on cancer cells have had the benefit of a longitudinal characterization as is afforded by our in vitro model system, which mimics the different stages of cervical carcinogenesis. We were able to show a progressive upregulation of PI3-kinase signalling during HPV-induced transformation by using the elevation of p-PKB/AKT as a reporter. Indeed, p-PBK/AKT was elevated in anchorage independent cells relative to HPV-immortalized cells. The expression levels of both PIK3CA and especially p-PKB/AKT in the latter cells were increased compared to primary keratinocytes (data not shown). This increased signalling functionally correlated with multiple attributes of transformed cells, such as cell growth, migration, and anchorage independent growth (Figure 3). The fact that specific silencing of PIK3CA using RNA interference affected each of the same transformed phenotypes in a negative manner, while PIK3CA overexpression increased proliferation (Figures 4 and 5) substantiates its function as an oncogene in cervical carcinogenesis, as has been suggested in previous studies [5, 8, 26].
Remarkably, our data also suggest a feedback effect in which PI3-kinase regulates HPV oncogene expression. HPV16 mRNA expression and hTERT mRNA were decreased after inhibition of PI3-kinase signalling (Figure 6). The downregulation of hTERT mRNA may be a direct result of reduced HPV expression, as E6 activates hTERT transcription . Thus, the reciprocal regulation between PIK3CA/PI3-kinase and viral oncogene expression acts in concert in maintaining the transformed phenotypes [32–34]. However, it is presently unknown whether the phenotypical effects of PI3-kinase inhibition seen in our model system reflect a direct consequence of E6/E7 repression or vice versa.
It has been shown that the oncoprotein E7 is able to activate PKB/AKT, which appeared to be dependent on its ability to bind and inactivate Rb gene family proteins [10, 35]. E7 may also maintain PKB/AKT in an active state, by binding and sequestering a known binding partner, the phosphatase PP2A, thereby inhibiting dephosphorylation of p-PKB/AKT . Active PKB/AKT is also known to activate MDM2, enhancing p53 degradation and elevating CDKs leading to pRb inactivation, further enhancing the established E6/E7 effect on p53 and pRb function [37, 38]. Moreover, activation of PKB/AKT by PI3-kinase can inhibit nuclear localization of p27kip1 and p21cip by phosphorylating the nuclear localization signal and hereby preventing nuclear transport and inducing proliferation, both of which are also affected by E7 overexpression [35, 39]. Here we show that besides the reported effect of HPVE7 on PI3-kinase activity, there is also a feed-back effect in that PI3-kinase can regulate HPV oncogene expression. From the present data it becomes clear that both mechanisms act in concert as the increased PI3-kinase activity appears essential for maintaining HPV oncogene expression. The need for a strict regulation of HPV expression, as also implicated in our previous study , suggests that yet other transformation-inducing host cell alterations contribute to the regulated expression of HPVE6/E7 as well as PI3-kinase signalling activity.
Organotypic raft cultures mimic a natural environment to evaluate the role of PI3-kinase signalling in the growth and differentiation of keratinocytes. Phosphorylation of PKB/AKT appeared to be tightly linked to differentiation, which is in agreement with a previous report showing upregulation of phosphorylated PKB/AKT in organotypic raft cultures of HPV16 expressing keratinocytes . Inhibiting PI3-kinase signalling by chemical inhibition and PIK3CA siRNA-mediated silencing during raft formation led to a dramatic reduction in p-PKB/AKT and severely hampered epithelial cell growth in both HPV16- and HPV18 immortalized cells (Figure 7). Proliferation marker Ki-67 remained detectable in residual epithelial cells, while differentiation marker CK10 was dramatically reduced. These results indicate that PIK3CA regulated p-PKB/AKT expression is important in squamous differentiation. This conclusion is in accordance with a previous report showing that mouse keratinocytes with active PKB/AKT have higher levels of differentiation markers Keratin 1, filaggrin and loricrin. Moreover, inhibition of PI3-kinase resulted in the specific death of differentiating keratinocytes . Similarly, treatment of primary esophageal keratinocytes with LY294002 resulted in an overall decrease in the number of basal keratinocytes and thickness of the epithelium . It has been suggested that PI3-kinase becomes important upon commitment of keratinocytes to differentiation, initiating the process and subsequently delivering the required survival signals . Based on the histology of our raft cultures, there were no apoptotic cells that are typified by highly condense and shrunken nuclei (Figure 7A). The thinning of the epithelium appears to stem from a severely curtailed proliferation. Hence, in organotypic raft culture systems, p-PKB/AKT appears to correlate with squamous differentiation and survival but not with proliferation, as is also evident from the lack of co-localization between p-PKB/AKT and Ki-67 (Figure 7). Also in a study by Menges et al  p-AKT is seen in differentiated non-proliferating BrdU negative cells. These observation are also in line with previous studies showing that AKT knock-out MEFs reduce BrdU incorporation by only 44-61% , indicating that AKT is not indispensible for proliferation, though the lack of it would slow down cell cycle progression at G1-S. We hypothesize that the balance in the dual character of PI3-kinase/PKB/AKT signalling shifts during the process of HPV-induced transformation towards tumor characteristics, rather than differentiation.
We have demonstrated that activation of the PI3-kinase/PKB/AKT pathway, in part resulting from increased PIK3CA expression, is functionally involved in HPV-mediated transformation in vitro. PIK3CA-mediated PI3-kinase signalling appeared essential in regulating proliferation, anchorage-independent growth, migration and importantly viral oncogene expression required to maintain the transformed phenotypes. Moreover, PIK3CA expression and PI3-kinase signalling emerged as critical regulators of epithelial growth and differentiation in organotypic raft cultures of HPV-immortalized cells. Hence, PIK3CA and/or the PI3-kinase/PKB/AKT pathway may provide suitable targets for therapeutic intervention in patients with HPV-induced carcinomas.
This research was supported by the VUmc Institute of Cancer and Immunology (V-ICI) and by USPHS grant CA83679 to LTC. We thank W Vos, D Claassen-Kramer, DM Schütze, L Bosch, and SM Wilting for excellent technical assistance and TM Roberts for providing the PIK3CA construct. We also thank AE Greijer and DAM Heideman for helpful discussion.
- zur Hausen H: Papillomavirus infections--a major cause of human cancers. Biochim Biophys Acta. 1996, 1288: F55-F78.PubMedGoogle Scholar
- von Knebel-Doeberitz M, Oltersdorf T, Schwarz E, Gissmann L: Correlation of modified human papilloma virus early gene expression with altered growth properties in C4-1 cervical carcinoma cells. Cancer Res. 1988, 48: 3780-3786.PubMedGoogle Scholar
- von Knebel-Doeberitz M, Rittmuller C, zur HH, Durst M: Inhibition of tumorigenicity of cervical cancer cells in nude mice by HPV E6-E7 anti-sense RNA. Int J Cancer. 1992, 51: 831-834. 10.1002/ijc.2910510527View ArticlePubMedGoogle Scholar
- Heselmeyer K, Macville M, Schrock E, Blegen H, Hellstrom AC, Shah K, Auer G, Ried T: Advanced-stage cervical carcinomas are defined by a recurrent pattern of chromosomal aberrations revealing high genetic instability and a consistent gain of chromosome arm 3q. Genes Chromosomes Cancer. 1997, 19: 233-240. 10.1002/(SICI)1098-2264(199708)19:4<233::AID-GCC5>3.0.CO;2-YView ArticlePubMedGoogle Scholar
- Ma YY, Wei SJ, Lin YC, Lung JC, Chang TC, Whang-Peng J, Liu JM, Yang DM, Yang WK, Shen CY: PIK3CA as an oncogene in cervical cancer. Oncogene. 2000, 19: 2739-2744. 10.1038/sj.onc.1203597View ArticlePubMedGoogle Scholar
- Wilting SM, Snijders PJ, Meijer GA, Ylstra B, van den Ijssel PR, Snijders AM, Albertson DG, Coffa J, Schouten JP, van de Wiel MA: Increased gene copy numbers at chromosome 20q are frequent in both squamous cell carcinomas and adenocarcinomas of the cervix. J Pathol. 2006, 209: 220-230. 10.1002/path.1966View ArticlePubMedGoogle Scholar
- Nicholson KM, Anderson NG: The protein kinase B/Akt signaling pathway in human malignancy. Cell Signal. 2002, 14: 381-395. 10.1016/S0898-6568(01)00271-6View ArticlePubMedGoogle Scholar
- Bertelsen BI, Steine SJ, Sandvei R, Molven A, Laerum OD: Molecular analysis of the PI3K-AKT pathway in uterine cervical neoplasia: frequent PIK3CA amplification and AKT phosphorylation. Int J Cancer. 2006, 118: 1877-1883. 10.1002/ijc.21461View ArticlePubMedGoogle Scholar
- Kohrenhagen N, Voelker HU, Schmidt M, Kapp M, Krockenberger M, Frambach T, Dietl J, Kammerer U: Expression of transketolase-like 1 (TKTL1) and p-Akt correlates with the progression of cervical neoplasia. J Obstet Gynaecol Res. 2008, 34: 293-300. 10.1111/j.1447-0756.2008.00749.xView ArticlePubMedGoogle Scholar
- Menges CW, Baglia LA, Lapoint R, McCance DJ: Human papillomavirus type 16 E7 up-regulates AKT activity through the retinoblastoma protein. Cancer Res. 2006, 66: 5555-5559. 10.1158/0008-5472.CAN-06-0499View ArticlePubMedGoogle Scholar
- Steenbergen RD, Walboomers JM, Meijer CJ, van der Raaij-Helmer EM, Parker JN, Chow LT, Broker TR, Snijders PJ: Transition of human papillomavirus type 16 and 18 transfected human foreskin keratinocytes towards immortality: activation of telomerase and allele losses at 3p, 10p, 11q and/or 18q. Oncogene. 1996, 19 (13): 1249-1257.Google Scholar
- Struijk L, van der ME, Kazem S, ter SJ, de Gruijl FR, Steenbergen RD, Feltkamp MC: Specific betapapillomaviruses associated with squamous cell carcinoma of the skin inhibit UVB-induced apoptosis of primary human keratinocytes. J Gen Virol. 2008, 89: 2303-2314. 10.1099/vir.0.83317-0View ArticlePubMedGoogle Scholar
- Zhao JJ, Liu Z, Wang L, Shin E, Loda MF, Roberts TM: The oncogenic properties of mutant p110alpha and p110beta phosphatidylinositol 3-kinases in human mammary epithelial cells. Proc Natl Acad Sci USA. 2005, 102: 18443-18448. 10.1073/pnas.0508988102PubMed CentralView ArticlePubMedGoogle Scholar
- Wilting SM, de Wilde J, Meijer CJ, Berkhof J, Yi Y, van Wieringen WN, Braakhuis BJ, Meijer GA, Ylstra B, Snijders PJ: Integrated genomic and transcriptional profiling identifies chromosomal loci with altered gene expression in cervical cancer. Genes Chromosomes Cancer. 2008, 47: 890-905. 10.1002/gcc.20590PubMed CentralView ArticlePubMedGoogle Scholar
- de Wilde J, Wilting SM, Meijer CJ, van de Wiel MA, Ylstra B, Snijders PJ, Steenbergen RD: Gene expression profiling to identify markers associated with deregulated hTERT in HPV-transformed keratinocytes and cervical cancer. Int J Cancer. 2008, 122: 877-888. 10.1002/ijc.23210View ArticlePubMedGoogle Scholar
- de Wilde J, De-Castro Arce J, Snijders PJ, Meijer CJ, Rosl F, Steenbergen RD: Alterations in AP-1 and AP-1 regulatory genes during HPV-induced carcinogenesis. Cell Oncol. 2008, 30: 77-87.PubMedGoogle Scholar
- Hesselink AT, van den Brule AJ, Groothuismink ZM, Molano M, Berkhof J, Meijer CJ, Snijders PJ: Comparison of three different PCR methods for quantifying human papillomavirus type 16 DNA in cervical scrape specimens. J Clin Microbiol. 2005, 43: 4868-4871. 10.1128/JCM.43.9.4868-4871.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Snijders PJ, Hogewoning CJ, Hesselink AT, Berkhof J, Voorhorst FJ, Bleeker MC, Meijer CJ: Determination of viral load thresholds in cervical scrapings to rule out CIN 3 in HPV 16, 18, 31 and 33-positive women with normal cytology. Int J Cancer. 2006, 119: 1102-1107. 10.1002/ijc.21956View ArticlePubMedGoogle Scholar
- Steenbergen RD, Kramer D, Braakhuis BJ, Stern PL, Verheijen RH, Meijer CJ, Snijders PJ: TSLC1 gene silencing in cervical cancer cell lines and cervical neoplasia. J Natl Cancer Inst. 2004, 96: 294-305. 10.1093/jnci/djh031View ArticlePubMedGoogle Scholar
- Steenbergen RD, Parker JN, Isern S, Snijders PJ, Walboomers JM, Meijer CJ, Broker TR, Chow LT: Viral E6-E7 transcription in the basal layer of organotypic cultures without apparent p21cip1 protein precedes immortalization of human papillomavirus type 16- and 18-transfected human keratinocytes. J Virol. 1998, 72: 749-757.PubMed CentralPubMedGoogle Scholar
- Vlahos CJ, Matter WF, Hui KY, Brown RF: A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem. 1994, 269: 5241-5248.PubMedGoogle Scholar
- Klingelhutz AJ, Foster SA, McDougall JK: Telomerase activation by the E6 gene product of human papillomavirus type 16. Nature. 1996, 380: 79-82. 10.1038/380079a0View ArticlePubMedGoogle Scholar
- Calautti E, Li J, Saoncella S, Brissette JL, Goetinck PF: Phosphoinositide 3-kinase signaling to Akt promotes keratinocyte differentiation versus death. J Biol Chem. 2005, 280: 32856-32865. 10.1074/jbc.M506119200View ArticlePubMedGoogle Scholar
- Janes SM, Ofstad TA, Campbell DH, Eddaoudi A, Warnes G, Davies D, Watt FM: PI3-kinase-dependent activation of apoptotic machinery occurs on commitment of epidermal keratinocytes to terminal differentiation. Cell Res. 2009, 19: 328-339. 10.1038/cr.2008.281PubMed CentralView ArticlePubMedGoogle Scholar
- Segrelles C, Moral M, Lara MF, Ruiz S, Santos M, Leis H, Garcia-Escudero R, Martinez-Cruz AB, Martinez-Palacio J, Hernandez P: Molecular determinants of Akt-induced keratinocyte transformation. Oncogene. 2006, 25: 1174-1185.View ArticlePubMedGoogle Scholar
- Zhang A, Maner S, Betz R, Angstrom T, Stendahl U, Bergman F, Zetterberg A, Wallin KL: Genetic alterations in cervical carcinomas: frequent low-level amplifications of oncogenes are associated with human papillomavirus infection. Int J Cancer. 2002, 101: 427-433. 10.1002/ijc.10627View ArticlePubMedGoogle Scholar
- Gao N, Flynn DC, Zhang Z, Zhong XS, Walker V, Liu KJ, Shi X, Jiang BH: G1 cell cycle progression and the expression of G1 cyclins are regulated by PI3K/AKT/mTOR/p70S6K1 signaling in human ovarian cancer cells. Am J Physiol Cell Physiol. 2004, 287: C281-C291. 10.1152/ajpcell.00422.2003View ArticlePubMedGoogle Scholar
- Shayesteh L, Lu Y, Kuo WL, Baldocchi R, Godfrey T, Collins C, Pinkel D, Powell B, Mills GB, Gray JW: PIK3CA is implicated as an oncogene in ovarian cancer. Nat Genet. 1999, 21: 99-102. 10.1038/5042View ArticlePubMedGoogle Scholar
- Sheng H, Shao J, Townsend CM, Evers BM: Phosphatidylinositol 3-kinase mediates proliferative signals in intestinal epithelial cells. Gut. 2003, 52: 1472-1478. 10.1136/gut.52.10.1472PubMed CentralView ArticlePubMedGoogle Scholar
- Zhao JJ, Gjoerup OV, Subramanian RR, Cheng Y, Chen W, Roberts TM, Hahn WC: Human mammary epithelial cell transformation through the activation of phosphatidylinositol 3-kinase. Cancer Cell. 2003, 3: 483-495. 10.1016/S1535-6108(03)00088-6View ArticlePubMedGoogle Scholar
- Lee CM, Fuhrman CB, Planelles V, Peltier MR, Gaffney DK, Soisson AP, Dodson MK, Tolley HD, Green CL, Zempolich KA: Phosphatidylinositol 3-kinase inhibition by LY294002 radiosensitizes human cervical cancer cell lines. Clin Cancer Res. 2006, 12: 250-256. 10.1158/1078-0432.CCR-05-1084View ArticlePubMedGoogle Scholar
- DeFilippis RA, Goodwin EC, Wu L, DiMaio D: Endogenous human papillomavirus E6 and E7 proteins differentially regulate proliferation, senescence, and apoptosis in HeLa cervical carcinoma cells. J Virol. 2003, 77: 1551-1563. 10.1128/JVI.77.2.1551-1563.2003PubMed CentralView ArticlePubMedGoogle Scholar
- Wells SI, Francis DA, Karpova AY, Dowhanick JJ, Benson JD, Howley PM: Papillomavirus E2 induces senescence in HPV-positive cells via pRB- and p21(CIP)-dependent pathways. EMBO J. 2000, 19: 5762-5771. 10.1093/emboj/19.21.5762PubMed CentralView ArticlePubMedGoogle Scholar
- Wells SI, Aronow BJ, Wise TM, Williams SS, Couget JA, Howley PM: Transcriptome signature of irreversible senescence in human papillomavirus-positive cervical cancer cells. Proc Natl Acad Sci USA. 2003, 100: 7093-7098. 10.1073/pnas.1232309100PubMed CentralView ArticlePubMedGoogle Scholar
- Charette ST, McCance DJ: The E7 protein from human papillomavirus type 16 enhances keratinocyte migration in an Akt-dependent manner. Oncogene. 2007, 26: 7386-7390. 10.1038/sj.onc.1210541View ArticlePubMedGoogle Scholar
- Pim D, Massimi P, Dilworth SM, Banks L: Activation of the protein kinase B pathway by the HPV-16 E7 oncoprotein occurs through a mechanism involving interaction with PP2A. Oncogene. 2005, 24: 7830-7838. 10.1038/sj.onc.1208935View ArticlePubMedGoogle Scholar
- Liang J, Slingerland JM: Multiple roles of the PI3K/PKB (Akt) pathway in cell cycle progression. Cell Cycle. 2003, 2: 339-345.View ArticlePubMedGoogle Scholar
- Zhou BP, Liao Y, Xia W, Zou Y, Spohn B, Hung MC: HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat Cell Biol. 2001, 3: 973-982. 10.1038/ncb1101-973View ArticlePubMedGoogle Scholar
- Westbrook TF, Nguyen DX, Thrash BR, McCance DJ: E7 abolishes raf-induced arrest via mislocalization of p21(Cip1). Mol Cell Biol. 2002, 22: 7041-7052. 10.1128/MCB.22.20.7041-7052.2002PubMed CentralView ArticlePubMedGoogle Scholar
- Van Tine BA, Kappes JC, Banerjee NS, Knops J, Lai L, Steenbergen RD, Meijer CL, Snijders PJ, Chatis P, Broker TR: Clonal selection for transcriptionally active viral oncogenes during progression to cancer. J Virol. 2004, 78: 11172-11186. 10.1128/JVI.78.20.11172-11186.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Oyama K, Okawa T, Nakagawa H, Takaoka M, Andl CD, Kim SH, Klein-Szanto A, Diehl JA, Herlyn M, El-Deiry W: AKT induces senescence in primary esophageal epithelial cells but is permissive for differentiation as revealed in organotypic culture. Oncogene. 2007, 26: 2353-2364. 10.1038/sj.onc.1210025PubMed CentralView ArticlePubMedGoogle Scholar
- Skeen JE, Bhaskar PT, Chen CC, Chen WS, Peng XD, Nogueira V, Hahn-Windgassen A, Kiyokawa H, Hay N: Akt deficiency impairs normal cell proliferation and suppresses oncogenesis in a p53-independent and mTORC1-dependent manner. Cancer Cell. 2006, 10: 269-280. 10.1016/j.ccr.2006.08.022View ArticlePubMedGoogle Scholar
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