Attenuation of PTEN increases p21 stability and cytosolic localization in kidney cancer cells: a potential mechanism of apoptosis resistance
- Pei-Yin Lin1,
- Susan P Fosmire2,
- See-Hyoung Park1,
- Jin-Young Park1,
- Shairaz Baksh3,
- Jaime F Modiano†2, 4 and
- Robert H Weiss†1, 5Email author
© Lin et al; licensee BioMed Central Ltd. 2007
Received: 02 January 2007
Accepted: 14 February 2007
Published: 14 February 2007
The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) tumor suppressor gene is frequently mutated or deleted in a wide variety of solid tumors, and these cancers are generally more aggressive and difficult to treat than those possessing wild type PTEN. While PTEN lies upstream of the phosphoinositide-3 kinase signaling pathway, the mechanisms that mediate its effects on tumor survival remain incompletely understood. Renal cell carcinoma (RCC) is associated with frequent treatment failures (~90% in metastatic cases), and these tumors frequently contain PTEN abnormalities.
Using the ACHN cell line containing wild type PTEN, we generated a stable PTEN knockdown RCC cell line using RNA interference. We then used this PTEN knockdown cell line to show that PTEN attenuation increases resistance to cisplatin-induced apoptosis, a finding associated with increased levels of the cyclin kinase inhibitor p21. Elevated levels of p21 result from stabilization of the protein, and they are dependent on the activities of phosphoinositide-3 kinase and Akt. More specifically, the accumulation of p21 occurs preferentially in the cytosolic compartment, which likely contributes to both cell cycle progression and resistance to apoptosis.
Since p21 regulates a decision point between repair and apoptosis after DNA damage, our data suggest that p21 plays a key role in mechanisms used by PTEN-deficient tumors to escape chemotherapy. This in turn raises the possibility to use p21 attenuators as chemotherapy sensitizers, an area under active continuing investigation in our laboratories.
The PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) gene encodes a dual lipid and tyrosine phosphatase that regulates signaling through the PI3K/Akt pathway , and acts as a tumor suppressor protein that is frequently mutated or deleted in human cancers. Studies have shown that mice heterozygous for PTEN develop spontaneous tumors[2, 3], and that conditional tissue-specific tissue disruption of PTEN leads to tumors in the affected tissues[4, 5]. Through its actions on multiple downstream signaling proteins, including but not limited to the PI3K/Akt pathway, PTEN has the capacity to affect a variety of cancer-relevant signaling cascades.
Germline mutations of PTEN occur in 80% of patients with Cowden syndrome, which is characterized by the occurrence of multiple non-cancerous hamartomas; in addition, these patients are at high risk for breast, thyroid, and endometrial carcinomas, as well as an increased risk of bladder and renal cell carcinoma (RCC). Consistent with these data, PTEN protein and gene expression have been variously described as reduced[7, 8], absent, mutated, or deleted  in human RCCs; a recent study demonstrated PTEN loss in 20% of RCCs and another study quoted an LOH of 27% in kidney cancer. Since RCC is a malignancy associated with frequent treatment failures when metastatic, and because RCC and other tumors lacking PTEN are often resistant to conventional chemotherapy[14, 15], the mechanism by which PTEN contributes to chemotherapy failure is of immediate clinical importance and may lead to new therapeutic options for patients with such cancers.
Cell cycle progression, both in normal and cancer cells, is finely regulated by the interplay between the cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors (CKIs), as well as by fluctuation in their levels at different points of the cell cycle (reviewed in ). The earliest described role of p21 was in cyclin/cdk inhibition[17, 18], but more recent data also has shown that p21 is involved in positive effects on cyclin/cdk activation[17, 19, 20] through its "assembly factor" function. In addition, p21 has been shown to be anti-apoptotic in many tissues, including cancer [22, 23], and, as such, has been suggested to be a target for cancer therapy. There are also reports of a role of p21 in inducing senescence, a mechanism which seems to protect against malignant transformation. We have previously shown that p21 is a prognostic marker in clear cell RCC (ccRCC) such that its elevated levels portend a poorer prognosis in patients who have metastatic ccRCC at diagnosis[26, 27].
While p21 is transcriptionally regulated by p53 (hence its function in DNA damage repair), the mechanisms that regulate the activity of p21 and its post-translational modification are less clear. A previous report demonstrated that p21 is phosphorylated by Akt, which leads to increased p21 stability as well as enhanced cell survival, and another report showed that cytoplasmic localization of p21 results from HER2/Neu activation of Akt with subsequent p21 phosphorylation. We have shown that p21 accumulates in the cytoplasm of actively growing cells  and that forced localization of p21 to the cytosolic compartment results in increased cell growth and resistance to apoptosis . Given the complex relationship between PTEN, phosphoinositide-3 kinase (PI3K), Akt, and p21, which are all signaling proteins involved in cell growth and apoptosis in cancer, we now address how PTEN deficiency influences p21. In this study, we demonstrate that, in an RCC cell line that retains wild type genes for PTEN and p53, knockdown of PTEN using RNA inhibition increases p21 stability, but not transcription. This event results in increased cytosolic localization of this protein, a property which, having been earlier shown to result in apoptosis resistance and cell cycle progression, may explain the chemoresistance of PTEN-deleted tumors.
Rapamycin decreases p21 in response to cisplatin-induced DNA damage
Inhibition of mTOR has been shown to decrease p21 in human non-small cell lung carcinoma cells, and, in fact, such attenuation of p21 is required for sensitization of these cells to apoptosis induced by DNA damage . A clinical trial using the rapamycin analog CCI-779 (an mTOR inhibitor) showed this compound has antitumor activity against RCC. We thus hypothesized that both the results from this trial and the chemotherapy resistance that is characteristic of PTEN-deficient RCC may be due, at least in part, to the anti-apoptotic function of p21.
Knockdown of PTEN increases steady-state levels of p21 and causes resistance to DNA-damage induced apoptosis
Stability of p21 is augmented in PTEN-knockdown cells
Inhibition of PI3K or Akt activation reverts p21 stability in PTEN-knockdown cells
Cytosolic localization of p21 is augmented in PTEN-downregulated cells
To confirm this finding, we performed subcellular localization of PTEN-knockdown and mutant shRNA control ACHN cells to determine whether p21 is directed more to the cytosol as a result of PTEN attenuation. In agreement with the immunofluorescence data, we found that the ratio of p21 in the cytosol as compared to the nucleus was markedly enhanced in PTEN-KD cells (Fig. 13b). Whether these findings are due to a generalized increase in cellular p21 with a stoichiometric redistribution of a proportion of the total p21 from the nucleus to the cytosol, or to a specific augmentation of cytosolic p21 through a separate signaling mechanism (such as alteration of the nuclear localization sequence), remains to be determined. However, augmentation of total p21 as well as specific increases in cytosolic p21 in PTEN-deficient cells may be an important mechanism through which PTEN-altered tumors acquire resistance to chemotherapy.
Many malignancies, including kidney cancer, are characterized by resistance to DNA-damaging chemotherapy. While the mechanisms of this effect remain incompletely understood, they appear to relate to activation of survival pathways that occur in the setting of DNA damage repair. For example, exposure to ionizing radiation causes DNA strand breaks leading to increases in p53, which in turn activates either the cell death (apoptosis) program, or alternatively, genes involved in cell cycle arrest and subsequent DNA repair (reviewed in ). Thus, tumors harboring p53 mutations (~50% of all human malignancies), fail to apoptose or growth arrest after DNA damage[47, 48] and may thus escape death upon exposure to chemotherapeutic agents; this can lead to chemotherapy failure. The cyclin kinase inhibitor p21 is induced by p53 in situations of DNA damage, and it likely plays a role in the decision pathways leading to apoptosis or DNA repair ; for this reason, not only has p21 been proposed as a target for chemotherapy sensitization, but also, the mechanisms by which p21 becomes activated have attracted considerable interest in the study of chemotherapy-resistant cancers.
p21 was initially characterized based on its function as an inhibitor of G1 cyclin kinases [17, 18], but after the discovery that p21 possesses pro-proliferative as well as anti-apoptotic  effects, research on this protein expanded to include to its roles in cancer progression and chemotherapy sensitivity. A putative relationship of p21 to PTEN was proposed after reports emerged that p21 is phosphorylated and stabilized by Akt[29, 39]. Our present findings reported here, that PTEN deficiency augments p21 stability and alters its subcellular localization so that it is situated more in the cytosolic compartment, may explain at least part of the observed chemotherapy resistance that is observed in PTEN-deficient tumor cell lines as well as in tumors derived from PTEN-knockout mice.
Other investigators have demonstrated a tissue-specific relationship between PTEN and p21. Using a Cre-Lox model of tissue-specific PTEN-deficiency, Yoo et al detected higher levels of p21 protein and transcripts in PTEN-deficient bladder tissue (but not prostate), a finding which they interpreted as a potential bladder-specific tumor suppression mechanism . Another group showed a similar phenomenon in PTEN-deficient prostate tissue, which they interpreted as confirming the activation of a p21-related senescence pathway. However, ours is the first study to report increased p21 levels and altered subcellular compartment localization in PTEN-knockdown cells, and to begin to elucidate a mechanism for this finding.
Our use of a kidney cancer cell line for these studies was prompted by the facts that (i) kidney cancer is exquisitely chemotherapy-resistant such that 5-year survival for the metastatic form of the disease is less than 10% , and (ii) decreased PTEN expression is frequently seen in this disease[8, 9]. In an earlier study, we found elevated levels of cytoplasmic p21 expression at time of surgery were associated with a worse prognosis in patients with metastatic ccRCC consistent with the data in the present study; assessment of PTEN and p21 status in a small set of ccRCC (N = 9) from the University of Colorado Hospital archives showed loss of PTEN and elevated levels of p21 staining in every tumor, as compared to unaffected kidney samples from age and sex matched controls (R. Weiss, J. Modiano, and K. Shroyer, unpublished observations).
Akt regulates the balance of apoptosis and cell survival by phosphorylating proteins crucial in apoptotic and anti-apoptotic mechanisms. Based on the fact that altered PTEN expression is often observed in RCC, deregulated Akt activation is likely one of the underlying mechanisms of RCC tumorigenesis. Use of the Akt inhibitor of the phosphatidylinositol ether lipid analogue class that we utilized in this study has been shown to cause apoptosis in various RCC lines which are characterized by elevated Akt activation . Among a variety of downstream targets of Akt, phosphorylation of Bad, an important pro-apoptotic protein, was prominently decreased when treated with the Akt inhibitor, possibly due to decreased Akt activity. In addition, expression of Bcl-XL, an anti-apoptotic protein, was decreased resulting in increased cell death. Consistent with these findings, our data suggest that pharmaceutical approaches to target Akt inhibition may be useful in designing more efficient therapeutic regimens for ccRCC.
The most recognized and universal response of a cell to DNA damage is activation of p53 transcription, followed by p53-mediated transcriptional activation. p53, the "guardian of the genome" is responsible for apoptosis of cells which cannot be repaired, and some of these biochemical events are mediated by p21. However, it has been described that PTEN may protect p53 from Mdm-2-mediated degradation, so it is possible that the observed increase in p21 levels is mediated via p53 or Mdm-2, or through a p53-independent mechanism.
The clinical relevance of our findings is supported by recent data indicating that p21 is involved in the mechanism of action of mTOR inhibitors. Beuvink et al showed that the mTOR inhibitor RAD001 sensitizes cells to DNA damage-induced apoptosis through inhibition of p21 translation. Our data suggests an explanation for the anti-tumor activity of mTOR inhibition in a clinical trial of RCC, especially in those RCCs with PTEN alterations, and these data support further trials of mTOR inhibitors in RCC and other HIF-activated cancers.
One possible means to sensitize cancer cells to DNA damage induced apoptosis would be to attenuate p21 prior to application of a DNA damaging agent in order to direct DNA damaged cells into the apoptotic (rather than the repair) pathway[23, 24]. Our reported successful use of phosphorothioated antisense p21 oligonucleotides in several cell lines, including renal mesangial cells [21, 23, 52, 53], could easily be extended to kidney disease in vivo, given the high transport of such oligonucleotides to kidney tubular epithelial cells when systemically administered. Experiments to test this possibility using both tissue culture and an animal model of RCC are currently underway in our laboratory.
In this study, we have found that knockdown of the commonly altered tumor suppressor gene PTEN results in increased stability and cytosolic localization of the pleiotropic cell cycle protein p21, both properties being historically associated with resistance to apoptosis. Whether this is a mechanism by which tumors escape chemotherapeutic death is a topic of active investigation in our laboratories; however, our findings suggest the use of novel p21-attenuating methods in combination with conventional chemotherapy in the treatment of chemotherapy-resistant kidney and other cancers.
Chemicals and reagents
The PI3K inhibitors LY-294,002 hydrochloride and wortmannin, and the proteasome inhibitors, N-acetyl-L-leucinyl-L-leucinal-L-norleucinal (LLnL), lactacystin and MG132, were purchased from Sigma-Aldrich (St. Louis, MO). The Akt inhibitor PIA5 was kindly provided by Dr. Phillip Dennis (Medical Oncology Branch, NCI). Anti-Akt, anti-phospho-Akt (Ser473), anti-mono-methyl Histone H3, anti-phospho-MDM2 (Ser 166), and anti-PTEN antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-p21 antibody was purchased from Upstate Biotechnology, Inc. (Charlottesville, VA). Anti-GAPDH antibody was from Chemicon-Millipore (Temecula, CA). Anti-phospho-threonine antibody was from Abcam (Cambridge, MA); and phospho-serine detection kit containing four different anti-phospho-serine antibodies, was from Calbiochem (San Diego, CA).
Design of vectors for RNA inhibition
We used the pSuper platform (OligoEngine, Seattle, WA) to clone PTEN short hairpin RNA molecules (shRNAs) as described. Small inhibitory RNA sequences were designed using a program similar to OligoEngine and Ambion (Austin, TX) online tools. Two sites that were common to both programs were selected, corresponding to positions 1,159 – 1,181 (AAGGCGTATACAG GAACAATATT) and 1,469 – 1,491 (AAGGCACAAGAGGCCCTAGATTT) from the human PTEN mRNA (Genbank accession # NM000314), where the ATG start site is at position 1,032. The oligonucleotides were cloned into the BglII/HindIII sites of the pSuper-puro vector as described . Specificity controls included the empty vector and a double base pair mutant for the 1,469 – 1,491 PTEN shRNA sequence (Fig. 1), where the resulting oligonucleotide did not show any positive matches for any mammalian sequence using basic blast (NCBI, National Library of Medicine, NIH). Stable cell lines integrating PTEN shRNA, mutant PTEN control, or empty pSuper-puro vector were selected in puromycin containing media.
Cells and cell lines
ACHN human RCC cells (CRL-1161) were obtained from ATCC (Manassas, VA) and maintained in MEM 1X media supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1 mM sodium pyruvate. For transfections, 1 × 106 cells were placed in a sterile cuvette with 4 μg of the corresponding pSuper vector + 1 μg of pEGFP-C vector (BD-Clontech, Mountain View, CA), and transfected using the AMAXA Nucleofector (Amaxa, Gaithersburg, MD) in solution V, program T20 following the manufacturer's instructions for transfection of adherent cells. Transfection efficiency was monitored microscopically based on the number of cells showing green fluorescence. Selection of stable cell lines was accomplished by adding 2.5 μg/ml puromycin to cells 24 hr after transfection. After 5 days, surviving cells were transferred to media containing 1 μg/ml puromycin, and the cells were maintained in that formulation throughout the duration of experiments. Retention of the transfected plasmids was routinely monitored by assessing green fluorescence in cells under selection, and by repeated assessment of PTEN levels using immunoblotting.
Cells were solubilized in lysis buffer (50 mM HEPES, 1% Triton X-100, 10 mM Na Pyrophosphate, 100 mM Na Fluoride, 4 mM EDTA) and equal protein quantities were electrophoresed and immunoblotted as previously described . The membranes were blocked in 5–10% non-fat dry milk or 1% BSA (for modification-state antibodies) for 1 h at room temperature, and probed with appropriate antibodies. Membranes were then probed with HRP-tagged anti-mouse or anti-rabbit IgG antibodies (Bio-Rad; Amersham) diluted 1:5,000 – 1:15,000 in 2.5–5% non-fat dry milk for 1 h at room temperature. Chemiluminescence was detected using enhanced ECL (Bio-Rad; Amersham Biosciences).
Data previously reported in reference were re-evaluated to determine the universality of the relationship between expression and localization of PTEN and p21. Briefly, archival tissues from canine melanomas were identified and 5 μm serial sections from paraffin-embedded blocks were mounted onto positively charged slides (Probe-on, Fisher Scientific, Pittsburgh, PA). Immunostaining was performed using a modified streptavidin- biotin complex method. Antigen retrieval was done by microwave heating for 6 minutes in a buffer of 0.1 M sodium citrate (pH 6). Samples were graded for intensity and percentage of positive cells, as well as for subcellular location of the relevant stain. Samples were considered negative when no staining was seen above that seen in negative controls. Staining that was fine, diffuse, and not always visible at low magnification (40–100-X), but was readily detectable in >30% of cells at high magnification (4,000-X) was considered weak. Staining that was prominent enough to be seen at low magnification in >30% of cells (and was verifiable as specific staining at high magnification) was considered moderate to strong. Samples were only considered positive when expression of these proteins was clearly detectable in melanocytic tumor cells (versus supporting stroma or inflammatory cells) based on the expression of S100a protein, Melan A, or neuron-specific enolase in serial sections from the tumors.
Protein stability assays
Approximately 2.5 × 105 cells were grown on 6-well tissue culture dishes in media containing 10% fetal bovine serum. Cells were treated with 20 μg/ml cycloheximide (Sigma-Aldrich) at 30 min interval up to a maximum of 2.5 h. Cells were collected and analyzed by immunoblotting.
Cells were grown on Lab-Tek multi-chamber slides (Nalge Nunc International, Rochester; NY, USA) and fixed in cold methanol for 10 min at -20°C. Fixed cells were rehydrated with PBS and blocked with 10% goat serum (Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Cells were incubated with anti-p21 antibody (1:50) for 30 min at room temperature, followed by a secondary goat anti-mouse IgG antibody (1:1000) conjugated to Alexa-Fluor 594 (Molecular Probes) for 30 min at room temperature. DAPI (0.1 μg/ml, 5 min at room temperature) was used for nuclear counterstaining. The LSM 5 Pascal confocal laser scanning microscope was used for imaging (Carl Zeiss; Thornwood, NY, USA).
The CaspACE assay kit (Promega, Madison, WI) was used to measure caspase-3 activity per the manufacturer's instructions. Briefly, 106 cells per 10 cm dish were treated as described in the text. Positive control cells were treated with 4 μM camptothecin. The cells were harvested, washed, and equal protein quantities were incubated with the DEVD-pNA caspace-3 substrate in Caspase Assay Buffer. The plates were covered with parafilm and incubated at 37°C overnight. Color development was measured at 405 nm.
Cells were grown in 100 cm dishes to 95% confluency followed by separation into nuclear and cytosolic fractions using the Panomics Nucelar extraction kit (Panomics, Fremont, CA) according to the manufacturer's instructions. Briefly, cells were washed with PBS twice and lysed in a buffer containing 100 mM HEPES, pH 7.9, 100 mM KCl, and 100 mM EDTA on ice for 10 minutes. Lysates were centrifuged at 15,000 × g for 3 min at 4°C to obtain cytosolic extracts. Soluble nuclear extracts were obtained by vortexing nuclear pellets in a buffer containing 100 mM HEPES, pH 7.9, 2 M NaCl, 5 mM EDTA, 50% glycerol, protease inhibitor cocktail and 100 mM DTT for 10 sec and then centrifuged at 15,000 × g for 5 min at 4°C. Protein concentrations in each fraction were determined using the BioRad protein assay. Relative enrichment of the cytosolic and nuclear fractions, respectively, were determined by immunoblotting with antibodies against GAPDH and Mono-Methyl Histone H3.
We thank Ashley Frazer-Abel and Silvia Oloris for assistance building the shRNA constructs. This work was supported by grant 1R21CA 91259-01A1 from NCI (RHW), grant RSG-02-173-01-LIB from the American Cancer Society (JFM), and grants 2025 and 422 from the AKC Canine Health Foundation (JFM), grant MOP-79494 from the CIHR (SB), grants from the VHL Family Alliance (PYL and RHW), Morris Animal Foundation (JFM and RHW), Dialysis Clinics, Inc (RHW), and the Monfort Family Foundation (JFM), and by directed donations from individuals to the Modiano lab at the University of Colorado Cancer Center.
- Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella BC, Ittmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R: PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science. 1997, 275: 1943-1947. 10.1126/science.275.5308.1943View ArticlePubMedGoogle Scholar
- Stambolic V, Tsao MS, Macpherson D, Suzuki A, Chapman WB, Mak TW: High incidence of breast and endometrial neoplasia resembling human Cowden syndrome in pten+/- mice. Cancer Res. 2000, 60: 3605-3611.PubMedGoogle Scholar
- Podsypanina K, Ellenson LH, Nemes A, Gu J, Tamura M, Yamada KM, Cordon-Cardo C, Catoretti G, Fisher PE, Parsons R: Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems. Proc Natl Acad Sci U S A. 1999, 96: 1563-1568. 10.1073/pnas.96.4.1563PubMed CentralView ArticlePubMedGoogle Scholar
- Wang S, Gao J, Lei Q, Rozengurt N, Pritchard C, Jiao J, Thomas GV, Li G, Roy-Burman P, Nelson PS, Liu X, Wu H: Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell. 2003, 4: 209-221. 10.1016/S1535-6108(03)00215-0View ArticlePubMedGoogle Scholar
- Yilmaz OH, Valdez R, Theisen BK, Guo W, Ferguson DO, Wu H, Morrison SJ: Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature. 2006, 441: 475-482. 10.1038/nature04703View ArticlePubMedGoogle Scholar
- Marsh DJ, Coulon V, Lunetta KL, Rocca-Serra P, Dahia PL, Zheng Z, Liaw D, Caron S, Duboue B, Lin AY, Richardson AL, Bonnetblanc JM, Bressieux JM, Cabarrot-Moreau A, Chompret A, Demange L, Eeles RA, Yahanda AM, Fearon ER, Fricker JP, Gorlin RJ, Hodgson SV, Huson S, Lacombe D, Eng C, : Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation. Hum Mol Genet. 1998, 7: 507-515. 10.1093/hmg/7.3.507View ArticlePubMedGoogle Scholar
- Lee JS, Kim HS, Kim YB, Lee MC, Park CS: Expression of PTEN in renal cell carcinoma and its relation to tumor behavior and growth. J Surg Oncol. 2003, 84: 166-172. 10.1002/jso.10289View ArticleGoogle Scholar
- Hara S, Oya M, Mizuno R, Horiguchi A, Marumo K, Murai M: Akt activation in renal cell carcinoma: contribution of a decreased PTEN expression and the induction of apoptosis by an Akt inhibitor. Ann Oncol. 2005, 16: 928-933. 10.1093/annonc/mdi182View ArticlePubMedGoogle Scholar
- Brenner W, Farber G, Herget T, Lehr HA, Hengstler JG, Thuroff JW: Loss of tumor suppressor protein PTEN during renal carcinogenesis. Int J Cancer. 2002, 99: 53-57. 10.1002/ijc.10303View ArticlePubMedGoogle Scholar
- Alimov A, Li C, Gizatullin R, Fredriksson V, Sundelin B, Klein G, Zabarovsky E, Bergerheim U: Somatic mutation and homozygous deletion of PTEN/MMAC1 gene of 10q23 in renal cell carcinoma. Anticancer Res. 1999, 19: 3841-3846.PubMedGoogle Scholar
- Sukosd F, Digon B, Fischer J, Pietsch T, Kovacs G: Allelic loss at 10q23.3 but lack of mutation of PTEN/MMAC1 in chromophobe renal cell carcinoma. Cancer Genet Cytogenet. 2001, 128: 161-163. 10.1016/S0165-4608(01)00413-7View ArticlePubMedGoogle Scholar
- Figlin RA, Seligson D, Wu H, Thomas G, Leppert JT, O'Toole T, Dukart G, Gibbons J, Belldegrun A, Pantuck AJ: Characterization of the mTOR pathway in renal cell carcinoma and its use in predicting patient selection for agents targeting this pathway. J Clin Oncol. 2005, 23: 4539-Google Scholar
- Eng C: PTEN: one gene, many syndromes. Hum Mutat. 2003, 22: 183-198. 10.1002/humu.10257View ArticlePubMedGoogle Scholar
- Zhou M, Gu L, Findley HW, Jiang R, Woods WG: PTEN reverses MDM2-mediated chemotherapy resistance by interacting with p53 in acute lymphoblastic leukemia cells. Cancer Res. 2003, 63: 6357-6362.PubMedGoogle Scholar
- Wendel HG, Malina A, Zhao Z, Zender L, Kogan SC, Cordon-Cardo C, Pelletier J, Lowe SW: Determinants of sensitivity and resistance to rapamycin-chemotherapy drug combinations in vivo. Cancer Res. 2006, 66: 7639-7646. 10.1158/0008-5472.CAN-06-0419PubMed CentralView ArticlePubMedGoogle Scholar
- Sherr CJ, Roberts JM: CDK inhibitors: positive and negative regulators of G1-phase progression. Genes and Dev. 1999, 13: 1501-1512.View ArticlePubMedGoogle Scholar
- Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D: p21 is a universal inhibitor of cyclin kinases. Nature. 1993, 366: 701-704. 10.1038/366701a0View ArticlePubMedGoogle Scholar
- Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ: The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993, 75: 805-816. 10.1016/0092-8674(93)90499-GView ArticlePubMedGoogle Scholar
- LaBaer J, Garrett MD, Stevenson LF, Slingerland JM, Sandhu C, Chou HS, Fattaey A, Harlow E: New functional activities for the p21 family of CDK inhibitors. Genes Dev. 1997, 11: 847-862.View ArticlePubMedGoogle Scholar
- Cheng M, Sexl V, Sherr CJ, Roussel MF: Assembly of cyclin D-dependent kinase and titration of p27Kip1 regulated by mitogen-activated protein kinase kinase (MEK1). Proc Natl Acad Sci U S A. 1998, 95. 10.1-1096.PubMed CentralView ArticlePubMedGoogle Scholar
- Weiss RH, Joo A, Randour C: p21Waf1/Cip1 is an assembly factor required for PDGF-induced vascular smooth muscle cell proliferation. J Biol Chem. 2000, 275: 10285-10290. 10.1074/jbc.275.14.10285View ArticlePubMedGoogle Scholar
- Weiss RH, Marshall D, Howard L, Corbacho AM, Cheung AT, Sawai ET: Suppression of breast cancer growth and angiogenesis by an antisense oligodeoxynucleotide to p21(Waf1/Cip1). Cancer Lett. 2003, 189: 39-48. 10.1016/S0304-3835(02)00495-0View ArticlePubMedGoogle Scholar
- Fan Y, Borowsky AD, Weiss RH: An antisense oligodeoxynucleotide to p21(Waf1/Cip1) causes apoptosis in human breast cancer cells. Mol Cancer Ther. 2003, 2: 773-782.PubMedGoogle Scholar
- Weiss RH: p21Waf1/Cip1 as a therapeutic target in breast and other cancers. Cancer Cell. 2003, 4: 425-429. 10.1016/S1535-6108(03)00308-8View ArticlePubMedGoogle Scholar
- Chen Z, Trotman LC, Shaffer D, Lin HK, Dotan ZA, Niki M, Koutcher JA, Scher HI, Ludwig T, Gerald W, Cordon-Cardo C, Pandolfi PP: Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature. 2005, 436: 725-730. 10.1038/nature03918PubMed CentralView ArticlePubMedGoogle Scholar
- Weiss RH, Borowsky AD, Seligson D, Lin PY, Dillard-Telm L, Belldegrun AS, Figlin RA, Pantuck AD: p21 is a prognostic marker for renal cell carcinoma: implications for novel therapeutic approaches. J Urol. 2007, 177: 63-68. 10.1016/j.juro.2006.08.073View ArticlePubMedGoogle Scholar
- Perroud B, Lee J, Valkova N, Dhirapong A, Lin PY, Fiehn O, Kultz D, Weiss RH: Pathway analysis of kidney cancer using proteomics and metabolic profiling. Mol Cancer. 2006, 5: 64- 10.1186/1476-4598-5-64PubMed CentralView ArticlePubMedGoogle Scholar
- Li Y, Jenkins CW, Nichols MA, Xiong Y: Cell cycle expression and p53 regulation of the cyclin-dependent kinase inhibitor p21. Oncogene. 1994, 9: 2261-2268.PubMedGoogle Scholar
- Li Y, Dowbenko D, Lasky LA: AKT/PKB phosphorylation of p21Cip1/WAF1 enhances protein stability of p21Cip1/WAF1 and promotes cell survival. J Biol Chem. 2002, 277: 11352-11361. 10.1074/jbc.M109062200View ArticlePubMedGoogle Scholar
- Zhou BP, Liao Y, Xia W, Spohn B, Lee MH, Hung MC: Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells. Nat Cell Biol. 2001, 3: 245-252. 10.1038/35060032View ArticlePubMedGoogle Scholar
- Ritt MG, Mayor J, Wojcieszyn J, Smith R, Barton CL, Modiano JF: Sustained nuclear localization of p21/WAF-1 upon growth arrest induced by contact inhibition. Cancer Lett. 2000, 158: 73-84. 10.1016/S0304-3835(00)00507-3View ArticlePubMedGoogle Scholar
- Dong Y, Chi SL, Borowsky AD, Fan Y, Weiss RH: Cytosolic p21Waf1/Cip1 increases cell cycle transit in vascular smooth muscle cells. Cell Signal. 2003, 16: 263-269. 10.1016/S0898-6568(03)00136-0.View ArticleGoogle Scholar
- Asada M, Yamada T, Ichijo H, Delia D, Miyazono K, Fukumuro K, Mizutani S: Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation. EMBO J. 1999, 18: 1223-1234. 10.1093/emboj/18.5.1223PubMed CentralView ArticlePubMedGoogle Scholar
- Beuvink I, Boulay A, Fumagalli S, Zilbermann F, Ruetz S, O'Reilly T, Natt F, Hall J, Lane HA, Thomas G: The mTOR inhibitor RAD001 sensitizes tumor cells to DNA-damage-induced apoptosis through inhibition of p21 translation. Cell. 2005, 120: 747-759. 10.1016/j.cell.2004.12.040View ArticlePubMedGoogle Scholar
- Atkins MB, Hidalgo M, Stadler WM, Logan TF, Dutcher JP, Hudes GR, Park Y, Liou SH, Marshall B, Boni JP, Dukart G, Sherman ML: Randomized phase II study of multiple dose levels of CCI-779, a novel mammalian target of rapamycin kinase inhibitor, in patients with advanced refractory renal cell carcinoma. J Clin Oncol. 2004, 22: 909-918. 10.1200/JCO.2004.08.185View ArticlePubMedGoogle Scholar
- Weiss RH, Lin PY: Kidney Cancer: Identification of Novel Targets for Therapy. Kidney Int. 2006, 69: 224-232. 10.1038/sj.ki.5000065View ArticlePubMedGoogle Scholar
- Nanus DM, Garino A, Milowsky MI, Larkin M, Dutcher JP: Active chemotherapy for sarcomatoid and rapidly progressing renal cell carcinoma. Cancer. 2004, 101: 1545-1551. 10.1002/cncr.20541View ArticlePubMedGoogle Scholar
- Koenig A, Bianco SR, Fosmire S, Wojcieszyn J, Modiano JF: Expression and significance of p53, rb, p21/waf-1, p16/ink-4a, and PTEN tumor suppressors in canine melanoma. Vet Pathol. 2002, 39: 458-472. 10.1354/vp.39-4-458View ArticlePubMedGoogle Scholar
- Rossig L, Jadidi AS, Urbich C, Badorff C, Zeiher AM, Dimmeler S: Akt-dependent phosphorylation of p21Cip1 regulates PCNA binding and proliferation in endothelial cells. Mol Cell Biol. 2001, 21: 5644-5657. 10.1128/MCB.21.16.5644-5657.2001PubMed CentralView ArticlePubMedGoogle Scholar
- Bloom J, Amador V, Bartolini F, DeMartino G, Pagano M: Proteasome-mediated degradation of p21 via N-terminal ubiquitinylation. Cell. 2003, 115: 71-82. 10.1016/S0092-8674(03)00755-4View ArticlePubMedGoogle Scholar
- Rousseau D, Cannella D, Boulaire J, Fitzgerald P, Fotedar A, Fotedar R: Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway. Oncogene. 1999, 18: 4313-4325. 10.1038/sj.onc.1202686View ArticlePubMedGoogle Scholar
- Blagosklonny MV, Wu GS, Omura S, el Deiry WS: Proteasome-dependent regulation of p21WAF1/CIP1 expression. Biochem Biophys Res Commun. 1996, 227: 564-569. 10.1006/bbrc.1996.1546View ArticlePubMedGoogle Scholar
- Raftopoulou M, Etienne-Manneville S, Self A, Nicholls S, Hall A: Regulation of cell migration by the C2 domain of the tumor suppressor PTEN. Science. 2004, 303: 1179-1181. 10.1126/science.1092089View ArticlePubMedGoogle Scholar
- Gills JJ, Holbeck S, Hollingshead M, Hewitt SM, Kozikowski AP, Dennis PA: Spectrum of activity and molecular correlates of response to phosphatidylinositol ether lipid analogues, novel lipid-based inhibitors of Akt. Mol Cancer Ther. 2006, 5: 713-722. 10.1158/1535-7163.MCT-05-0484View ArticlePubMedGoogle Scholar
- Winters ZE, Leek RD, Bradburn MJ, Norbury CJ, Harris AL: Cytoplasmic p21Waf1/Cip1 expression is correlated with HER-2/neu in breast cancer and is an independent predictor of prognosis. Breast Cancer Res. 2003, 5: 242-249. 10.1186/bcr654.View ArticleGoogle Scholar
- Winters ZE, Hunt NC, Bradburn MJ, Royds JA, Turley H, Harris AL, Norbury CJ: Subcellular localisation of cyclin B, Cdc2 and p21(WAF1/CIP1) in breast cancer. association with prognosis. Eur J Cancer. 2001, 37: 2405-2412. 10.1016/S0959-8049(01)00327-6View ArticlePubMedGoogle Scholar
- Kastan MB, Onyekwere O, Sidransky D, Vogelstein B, Craig RW: Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991, 51: 6304-6311.PubMedGoogle Scholar
- Kuerbitz SJ, Plunkett BS, Walsh WV, Kastan MB: Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci U S A. 1992, 89: 7491-7495. 10.1073/pnas.89.16.7491PubMed CentralView ArticlePubMedGoogle Scholar
- Wu H, Goel V, Haluska FG: PTEN signaling pathways in melanoma. Oncogene. 2003, 22: 3113-3122. 10.1038/sj.onc.1206451View ArticlePubMedGoogle Scholar
- Yoo LI, Liu DW, Le Vu S, Bronson RT, Wu H, Yuan J: Pten deficiency activates distinct downstream signaling pathways in a tissue-specific manner. Cancer Res. 2006, 66: 1929-1939. 10.1158/0008-5472.CAN-05-1986View ArticlePubMedGoogle Scholar
- Freeman DJ, Li AG, Wei G, Li HH, Kertesz N, Lesche R, Whale AD, Martinez-Diaz H, Rozengurt N, Cardiff RD, Liu X, Wu H: PTEN tumor suppressor regulates p53 protein levels and activity through phosphatase-dependent and -independent mechanisms. Cancer Cell. 2003, 3: 117-130. 10.1016/S1535-6108(03)00021-7View ArticlePubMedGoogle Scholar
- Weiss RH, Randour CJ: Attenuation of matrix protein secretion by antisense oligodeoxynucleotides to the cyclin kinase inhibitor p21(Waf1/Cip1). Atherosclerosis. 2002, 161: 105-112. 10.1016/S0021-9150(01)00628-1View ArticlePubMedGoogle Scholar
- Fan YP, Weiss RH: Exogenous attenuation of p21(Waf1/Cip1) decreases mesangial cell hypertrophy as a result of hyperglycemia and IGF-1. J Am Soc Nephrol. 2004, 15: 575-584. 10.1097/01.ASN.0000114557.75244.5FView ArticlePubMedGoogle Scholar
- Rappaport J, Hanss B, Kopp JB, Copeland TD, Bruggeman LA, Coffman TM, Klotman PE: Transport of phosphorothioate oligonucleotides in kidney: implications for molecular therapy. Kidney Int. 1995, 47: 1462-1469.View ArticlePubMedGoogle Scholar
- Baksh S, Tommasi S, Fenton S, Yu VC, Martins LM, Pfeifer GP, Latif F, Downward J, Neel BG: The tumor suppressor RASSF1A and MAP-1 link death receptor signaling to Bax conformational change and cell death. Mol Cell. 2005, 18: 637-650. 10.1016/j.molcel.2005.05.010View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.