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Protective Function of p27KIP1 against Apoptosis in Small Cell Lung Cancer Cells in Unfavorable Microenvironments

2001; Elsevier BV; Volume: 158; Issue: 1 Linguagem: Inglês

10.1016/s0002-9440(10)63947-8

1525-2191

Akira Masuda, Hirotaka Osada, Yasushi Yatabe, Ken‐ichi Kozaki, Yoshio Tatematsu, Takao Takahashi, Toyoaki Hida, Takashi Takahashi, Takashi Takahashi,

Cancer Research and Treatments

A previous study of ours unexpectedly found that in contrast to frequent reductions in non-small cell lung cancer, high expression of the p27KIP1 cyclin-dependent kinase (CDK) inhibitor was retained in virtually all small cell lung cancers (SCLCs), suggesting the possibility of high expression of nonfunctional p27KIP1 in this virulent tumor. The study presented here, however, shows that p27KIP1in SCLC biochemically functions as a CDK inhibitor, clearly showing induction apparently associated with G1/G0 arrest and efficient binding to and inhibition of the cyclin E-CDK2 complex. Interestingly, induction of p27KIP1 seems to confer on SCLC cells the ability to survive under culture conditions unfavorable for cell growth such as a lack of nutrients and hypoxia. Subsequent experiments manipulating p27KIP1 levels by using a sense p27KIP1 expression construct or an antisense oligonucleotide supported this notion. These observations suggest that high expression of p27KIP1 in vivo may favor the survival of SCLC by preventing apoptosis in a microenvironment unfavorable for cell proliferation. A previous study of ours unexpectedly found that in contrast to frequent reductions in non-small cell lung cancer, high expression of the p27KIP1 cyclin-dependent kinase (CDK) inhibitor was retained in virtually all small cell lung cancers (SCLCs), suggesting the possibility of high expression of nonfunctional p27KIP1 in this virulent tumor. The study presented here, however, shows that p27KIP1in SCLC biochemically functions as a CDK inhibitor, clearly showing induction apparently associated with G1/G0 arrest and efficient binding to and inhibition of the cyclin E-CDK2 complex. Interestingly, induction of p27KIP1 seems to confer on SCLC cells the ability to survive under culture conditions unfavorable for cell growth such as a lack of nutrients and hypoxia. Subsequent experiments manipulating p27KIP1 levels by using a sense p27KIP1 expression construct or an antisense oligonucleotide supported this notion. These observations suggest that high expression of p27KIP1 in vivo may favor the survival of SCLC by preventing apoptosis in a microenvironment unfavorable for cell proliferation. Lung cancer is among the leading causes of cancer death in economically developed countries. It is classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), which exhibit considerably distinct clinicopathological, biological, and molecular genetic characteristics.1Minna JD Sekido Y Fong KM Gazdar AF Cancer of the lung.in: DeVita VT Hellman S Rosenberg SA Cancer Principles and Practice of Oncology. Lippincott-Raven, Philadelphia1997: 849-949Google Scholar, 2Travis WD Linder J Mackay B Classification, histology, cytology, and electron microscopy.in: Pass HI Mitchell JB Johnson DH Turisi AT Lung Cancer: Principles and Practice. Lippincott-Raven, Philadelphia1996: 361-395Google Scholar Prognosis of SCLC cases is generally worse than that of NSCLC patients because of the former’s propensity for earlier aggressive growth and metastasis. Recent studies on cell cycle control in relation to carcinogenesis have revealed that cyclin-dependent kinase (CDK) inhibitors, which negatively regulate the ordered activation of cyclin-CDK complexes during the cell cycle, may play a role as tumor suppressor genes.3Sherr CJ Cancer cell cycles.Science. 1996; 274: 1672-1677Crossref PubMed Scopus (5032) Google Scholar For example, genetic and epigenetic inactivation of the p16 gene has been reported in various human cancers including NSCLC,4Kamb A Gruis NA Weaver-Feldhaus J Liu Q Harshman K Tavtigian SV Stockert E Day III, RS Johnson BE Skolnick MH A cell cycle regulator potentially involved in genesis of many tumor types.Science. 1994; 264: 436-440Crossref PubMed Scopus (2866) Google Scholar, 5Nobori T Miura K Wu DJ Lois A Takabayashi K Carson D Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers.Nature. 1994; 368: 753-756Crossref PubMed Scopus (1785) Google Scholar, 6Merlo A Herman JG Mao L Lee DJ Gabrielson E Burger PC Baylin SB Sidransky D 5′ CpG island methylation is associated with transcriptional silencing of the tumor suppressor p16/CDKN2/MTS1 in human cancer.Nat Med. 1995; 1: 686-692Crossref PubMed Scopus (1890) Google Scholar, 7Washimi O Nagatake M Osada H Ueda R Koshikawa T Seki T Takahashi T Takahashi T In vivo occurrence of p16 (MTS1) and p15 (MTS2) alterations preferentially in non-small cell lung cancers.Cancer Res. 1995; 55: 514-517PubMed Google Scholar whereas a previous report of ours suggested the potential involvement of the imprinted p57KIP2 gene in the development of lung cancers because of selective loss of the active maternal allele.8Kondo M Matsuoka S Uchida K Osada H Nagatake M Takagi K Harper JW Takahashi T Elledge SJ Takahashi T Selective maternal-allele loss in human lung cancers of maternally expressed p57KIP2 gene at 11p15.5.Oncogene. 1996; 12: 1365-1368PubMed Google Scholar p27KIP1, a member of the CIP/KIP CDK inhibitor family, is expressed in large amounts in quiescent cells and declines before cellular proliferation in response to mitogenic signals.9Coats S Flanagan M Nourse J Roberts JM Requirement of p27KIP1 for restriction point control of the fibroblast cell cycle.Science. 1996; 272: 877-880Crossref PubMed Scopus (655) Google Scholar Althoughp27KIP1 may also function as a potential tumor suppressor gene, virtually no somatic mutations have been reported in human neoplasms thus far.10Ponce-Castañeda MV Lee M-H Latres E Polyak K Lacombe L Montgomery K Mathew S Krauter K Sheinfeld J Massague J Cordon-Cardo C p27KIP1: chromosomal mapping to 12p12-12p13.1 and absence of mutations in human tumors.Cancer Res. 1995; 55: 1211-1214PubMed Google Scholar, 11Kawamata N Morosetti R Miller CW Park D Spirin KS Nakamaki T Takeuchi S Hatta Y Simpson J Wilcyznski S Lee YY Bartram CR Koeffler HP Molecular analysis of the cyclin-dependent kinase inhibitor gene p27/Kip1 in human malignancies.Cancer Res. 1995; 55: 2266-2269PubMed Google Scholar We previously reported that significantly reduced p27KIP1expression is frequent in NSCLC and associated with shortened patient survival. On the other hand, high p27KIP1expression was unexpectedly detected in virtually all cases of SCLC, which is known to be the most virulent type of human lung cancer.12Yatabe Y Masuda A Koshikawa T Nakamura S Kuroishi T Osada H Takahashi T Mitsudomi T Takahashi T p27KIP1 in human lung cancers: differential changes in small cell and non-small cell carcinomas.Cancer Res. 1998; 58: 1042-1047PubMed Google Scholar This observation could not be easily reconciled with this inhibitor’s function as a negative regulator of the cell cycle, and raised the possibility that highly expressed p27KIP1 in SCLC may be in a nonfunctional state because of multiple genetic defects such as myc gene amplification and Rb inactivation. In this connection, Vlach and colleagues13Vlach J Hennecke S Alevizopoulos K Conti D Amati B Growth arrest by the cyclin-dependent kinase inhibitor p27KIP1 is abrogated by c-Myc.EMBO J. 1996; 15: 6595-6604Crossref PubMed Scopus (299) Google Scholar reported that c-myc could abrogate growth arrest by sequestering p27KIP1 in a form that cannot bind the cyclin E-CDK2 complex. Alternatively, the almost invariably occurring inactivation of Rb, which is negatively regulated by the cyclin-CDK complexes, might allow SCLC cells to proliferate in the presence of high p27KIP1expression. In the study presented here, we examined the regulation and functions of highly expressed p27KIP1 in SCLC as well as its biological consequences. We were able to show that p27KIP1 can be induced by unfavorable changes in the cellular microenvironment such as nutrient insufficiency and low oxygen pressure, that it is biochemically functional as a CDK inhibitor in apparent association with G1/G0 arrest, and that it confers on SCLC cells the ability to escape from apoptosis under conditions unfavorable for cell growth. Therapeutic implications of these observations are also discussed. RPMI 1640 powder and the selectamine kit for preparation of the isoleucine-free medium were purchased from Life Technologies, Inc. (Gaithersburg, MD) and fetal calf serum (FCS) from JRH Bioscience (Lenexa, KS). The human SCLC cell lines, ACC-LC-35, -48, -49, -51, and -67, were established in our laboratory14Sekido Y Takahashi T Mäkelä TP Obata Y Ueda R Hida T Hibi K Shimokata K Alitalio K Takahashi T Complex intrachromosomal rearrangement in the process of amplification of the L-myc gene in small-cell lung cancer.Mol Cell Biol. 1992; 12: 1747-1754PubMed Google Scholar and kept in a plastic flask for suspension culture (Sumilon MS-2005R; Sumitomo Bakelite, Tokyo, Japan). Twice a week the cell lines were supplied with RPMI 1640 medium supplemented with 5% FCS. Molecular genetic studies have demonstrated the inactivation of the Rb and p53 genes and amplification of the L-myc gene in ACC-LC-48 and ACC-LC-49. For immunohistochemical, biochemical, and fluorescence-activated cell sorting (FACS) analyses, 5 × 105 cells were inoculated in 35-mm dishes for suspension culture (Falcon 1008; Becton Dickinson, Bedford, MA) with either complete or isoleucine-free RPMI 1640 medium supplemented with dialyzed 5% FCS and 0.65% methylcellulose (4000 cps; Katayama Chemical, Osaka, Japan) and incubated at 37°C for given periods. Hypoxic conditions were generated with an anaerobic sealed bag and an anaerobic culture system consisting of oxygen absorber, CO2 generator, and oxygen indicator (FX-4; Cosmo Bio Co., Ltd., Tokyo, Japan). With this system, it was possible to keep and monitor the oxygen concentration at 80% of the cells had also been heavily stained with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method by day 3 (data not shown). In contrast, there were very few apoptotic cells in the complete medium (Figure 4A). Putting these results together suggests that induction of p27KIP1 may play a role in the protection of SCLC cells from apoptosis under culture conditions unfavorable for cell growth. SCLCs, which feature poor vascularity relative to their rich cellularity, are thought to be hypoxic in vivo and tumor cell hypoxia is considered as one of the causes of tumor treatment resistance.19Urtasun RC Parliament MB McEwan AJ Mercer JR Mannan RH Wiebe LI Morin C Chapman JD Measurement of hypoxia in human tumors by non-invasive spect imaging of iodoazomycin arabinoside.Br J Cancer. 1996; 74: S209-S212Google Scholar, 20Urtasun RC Palmer M Kinney B Belch A Hewitt J Hanson J Intervention with the hypoxic tumor cell sensitizer etanidazole in the combined modality treatment of limited stage small-cell lung cancer. A one-institution study.Int J Radiat Oncol Biol Phys. 1998; 40: 337-342Abstract Full Text PDF PubMed Scopus (20) Google Scholar We therefore examined whether another unfavorable microenvironment, hypoxia, can induce p27KIP1 expression in SCLC cell lines. Western blot analysis showed that p27KIP1 expression was clearly increased in ACC-LC-48 within 24 hours when cells were placed in a hypoxic environment, resulting in the expression of p27KIP1 at a level similar to that observed under incomplete nutrient conditions (Figure 5). ACC-LC-48 cells with increased p27KIP1 expression were found to be resistant to apoptosis under hypoxic conditions. In contrast, an increase in apoptotic cells in a hypoxic environment was evident in ACC-LC-49, which did not show a marked induction of p27KIP1. These results offer further support for the suggestion that induction of p27KIP1 may play a role in the protection of SCLC cells from apoptosis under culture conditions unfavorable for cell growth such as insufficient nutrients and lack of oxygen. To further examine the relationship between anti-apoptotic activity and induction of p27KIP1 in SCLC, a human p27KIP1 cDNA was introduced together with a GFP (green fluorescence protein) expression vector into ACC-LC-49 with a constitutive low expression of p27KIP1 even in the isoleucine-free medium. Two days after transfection, the cells were transferred to either the complete or the isoleucine-free medium, and further incubated for 2 days. As shown in Figure 6, the introduction of p27KIP1 resulted in a significant increase in surviving GFP-positive cells in the isoleucine-free medium, whereas there was no such augmentation when they were cultured in the complete medium. A complementary experiment was performed using ACC-LC-48 cells that contain highly inducible p27KIP1. Either an antisense or mismatch oligonucleotide was transfected to ACC-LC-48. After a 1-day incubation of the transfected cells in the complete medium, they were transferred into either the complete or isoleucine-free medium. A significant decrease in the number of nonapoptotic surviving cells was observed in the isoleucine-free medium but not in the complete medium, in association with the reduction in p27KIP1 expression resulting from the antisense p27KIP1 oligonucleotide treatment (Figure 7). Introduction of the mismatch oligonucleotide did not produce such effects. Together, these observations suggest that p27KIP1 may play a role in the protection of SCLC cells from apoptosis under unfavorable conditions for cell growth. It should be noted that our preliminary experiments showed NSCLC cell lines to be rather refractory to the induction of apoptosis after the introduction of antisense p27KIP1 even in the isoleucine-free medium. This suggests that p27KIP1-dependent resistance to apoptosis may, to certain extent, be specific to SCLC cells (unpublished observation). Our previous immunohistochemical examination of p27KIP1 expression in human lung cancers unexpectedly showed a marked increase in virtually all SCLC cases. The study reported here clearly demonstrates that p27KIP1 expression in SCLC cell lines is normally regulated by the extracellular microenvironment and is not constitutively expressed in a tissue culture or in transplanted tumors. We were also able to show that the cyclin E- or CDK2-associated histone H1-kinase activity is markedly inhibited in SCLC cells under culture conditions unfavorable for cell growth, presumably because of a significant increase of p27KIP1 in the cyclin-CDK complexes. Although SCLC is known to frequently carry c -myc amplification and overexpression,1Minna JD Sekido Y Fong KM Gazdar AF Cancer of the lung.in: DeVita VT Hellman S Rosenberg SA Cancer Principles and Practice of Oncology. Lippincott-Raven, Philadelphia1997: 849-949Google Scholar it was previously reported that in an experimental system using Rat1 cells, c-myc could induce abrogation of growth arrest by sequestering p27KIP1 from the cyclin E-CDK2 complex.13Vlach J Hennecke S Alevizopoulos K Conti D Amati B Growth arrest by the cyclin-dependent kinase inhibitor p27KIP1 is abrogated by c-Myc.EMBO J. 1996; 15: 6595-6604Crossref PubMed Scopus (299) Google Scholar The present findings, however, suggest that p27KIP1 is functional as a cell cycle regulator not only in normal lung epithelial cells12Yatabe Y Masuda A Koshikawa T Nakamura S Kuroishi T Osada H Takahashi T Mitsudomi T Takahashi T p27KIP1 in human lung cancers: differential changes in small cell and non-small cell carcinomas.Cancer Res. 1998; 58: 1042-1047PubMed Google Scholar but also in SCLC cells. It is nevertheless possible that high p27KIP1 expression in SCLC may stoichiometrically titrate out the sequestering action of c-myc and that such changes in the delicate balance might consequently induce growth arrest despite myc overexpression. One of the characteristic histological features of SCLC tumors is the presence of patches of massive cell death, suggesting their susceptibility to apoptosis.2Travis WD Linder J Mackay B Classification, histology, cytology, and electron microscopy.in: Pass HI Mitchell JB Johnson DH Turisi AT Lung Cancer: Principles and Practice. Lippincott-Raven, Philadelphia1996: 361-395Google Scholar SCLC shows very rich cellularity together with relatively poor vascularity, which is possibly an architectural prerequisite for insufficient blood supply and low oxygen pressure.19Urtasun RC Parliament MB McEwan AJ Mercer JR Mannan RH Wiebe LI Morin C Chapman JD Measurement of hypoxia in human tumors by non-invasive spect imaging of iodoazomycin arabinoside.Br J Cancer. 1996; 74: S209-S212Google Scholar, 20Urtasun RC Palmer M Kinney B Belch A Hewitt J Hanson J Intervention with the hypoxic tumor cell sensitizer etanidazole in the combined modality treatment of limited stage small-cell lung cancer. A one-institution study.Int J Radiat Oncol Biol Phys. 1998; 40: 337-342Abstract Full Text PDF PubMed Scopus (20) Google Scholar Our study suggests that p27KIP1 can confer on SCLC cells resistance to apoptosis because of an unfavorable microenvironment. It is possible that expression of p27KIP1 in SCLC cells in vivo may be a reflection of unfavorable growth conditions such as insufficient nutrients and low tissue oxygen pressure as shown in vitro in our study. Thus, p27KIP1 might function as a remaining gatekeeper in SCLC cells, which show neuroendocrine differentiation, harbor multiple genetic defects including invariable Rb inactivation,21Harbour JW Lai S-L Whang-Peng J Gazdar AF Minna JD Kaye FJ Abnormalities in structure and expression of the human retinoblastoma gene in SCLC.Science. 1988; 241: 353-357Crossref PubMed Scopus (834) Google Scholar and very frequent p53 mutations.22Takahashi T Nau MM Chiba I Birrer MJ Rosenberg RK Vinocour M Levitt M Pass H Gazdar AF Minna JD p53: a frequent target for genetic abnormalities in lung cancer.Science. 1989; 246: 491-494Crossref PubMed Scopus (1235) Google Scholar In this regard, it has been suggested that p27KIP1 may play a part in protecting cells and tissues from inflammatory injury by acting as a safeguard against excessive cell proliferation and apoptosis.23Ophascharoensuk V Fero ML Hughes J Roberts JM Shankland SJ The cyclin-dependent kinase inhibitor p27Kip1 safeguards against inflammatory injury.Nat Med. 1998; 4: 575-580Crossref PubMed Scopus (181) Google Scholar A similar protective function of p27KIP1 was also implicated in growth factor-deprived fibroblasts from p27KIP1 null mice.24Hiromura K Pippin JW Fero ML Roberts JM Shankland SJ Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27Kip1.J Clin Invest. 1999; 103: 597-604Crossref PubMed Scopus (208) Google Scholar In addition, it may be of interest that a previous study of ours suggested a possible link between high expression of p27KIP1 and neuroendocrine differentiation of SCLC cells, because prominent apoptosis was reported to be elicited in neuronal cells of Ink4d- and p27KIP1-double-knockout mice,25Zingy F Cunningham JJ Sherr CJ Jogal S Smeyne RJ Roussel MF Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin-dependent kinases.Proc Natl Acad Sci USA. 1999; 96: 13462-13467Crossref PubMed Scopus (154) Google Scholar which can be viewed as reminiscent of p27KIP1-deprived SCLC cells carrying Rb mutations in terms of cell cycle control. SCLC is generally very sensitive to chemotherapeutic agents and irradiation, but recurrence is quite frequent, indicating inadequate eradication of tumor cells by currently available therapeutic modalities.26Ihde DC Pass HI Glatstein E Small cell lung cancer.in: DeVita VT Hellman S Rosenberg SA Cancer Principles and Practice of Oncology. Lippincott-Raven, Philadelphia1997: 911-949Google Scholar The existence of quiescent cells such as hypoxic cells is considered to be one of the multifactorial causes of tumor treatment resistance. For this reason, the results presented here warrant further studies aiming at a thorough elimination of the residual SCLC cells by forced reduction of the p27KIP1 expression level. Potential strategies for such novel therapeutics may well include p27KIP1antisense oligonucleotides and viral vectors as well as agents that selectively enhance degradation of p27KIP1. It is also worth noting that an association between induction of p27KIP1 and drug resistance was shown in a murine mammary tumor cell line and that p27KIP1antisense oligonucleotides in combination with conventional anticancer drugs enhanced apoptosis.17St Croix B Flørenes VA Rak JW Flanagan M Bhattacharya N Slingerland JM Kerbel RS Impact of the cyclin-dependent kinase inhibitor p27Kip1 on resistance of tumor cells to anticancer agents.Nat Med. 1996; 2: 1204-1210Crossref PubMed Scopus (303) Google Scholar, 27St Croix B Kerbel RS Cell adhesion and drug resistance in cancer.Curr Opin Oncol. 1997; 9: 549-556Crossref PubMed Scopus (115) Google Scholar Such a combinatorial treatment may prove to be useful as an adjunct therapy for the treatment of SCLC.