Eileen Friedman

The Cyclin-dependent Kinase Inhibitor p27Kip1 Is Stabilized in G0 by Mirk/dyrk1B Kinase

Elevated levels of the cyclin-dependent kinase (CDK) inhibitor p27 block the cell in G0/G1 until mitogenic signals activate G1 cyclins and initiate proliferation. Post-translational regulation of p27 by different phosphorylation events is critical in allowing cells to proceed through the cell cycle. We now demonstrate that the arginine-directed kinase, Mirk/dyrk1B, is maximally active in G0 in NIH3T3 cells, when it stabilizes p27 by phosphorylating it at Ser-10. The phospho-mimetic mutant p27-S10D was more stable, and the non-phosphorylatable mutant p27-S10A was less stable than wild-type when expressed in G0-arrested cells. Following phosphorylation by Mirk, p27 remains a functional CDK inhibitor, capable of binding to CDK2. Mirk did not induce the translocation of p27 from the nucleus in G0, but instead co-localized with nuclear p27. Depletion of Mirk by RNA interference decreased the phosphorylation of p27 at Ser-10 and the stability of endogenous p27. RNAi to Mirk increased cell entry from G0 into G1 as shown by increased expression of proliferating cell nuclear antigen and decreased expression of p27. These data suggest a model in which Mirk increases the amount of nuclear p27 by stabilizing it during G0 when Mirk is most abundant. Mitogen stimulation then causes cells to enter G1, reduces Mirk levels (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354), and initiates the translocation of p27 to the cytoplasm. In addition, depletion of Mirk by RNAi in postmitotic C2C12 myoblasts decreased protein but not mRNA levels of p27, suggesting that stabilization of p27 by Mirk also occurs during differentiation.

Oncogenic Ki-ras but Not Oncogenic Ha-rasBlocks Integrin β1-Chain Maturation in Colon Epithelial Cells

Human colorectal tumors commonly contain mutations in Ki-ras but rarely, if ever, in Ha-ras. The selectivity for Ki-ras mutations in this tumor was explored using the HD6-4 colon epithelial cell line which contains no ras mutations. After adhesion to an extracellular matrix, HD6-4 cells polarize into columnar goblet cells with distinct apical and basal regions. Stable HD6-4 transfectants were made with mini-gene constructs of the oncogenic cellular Ki-ras4BG12V gene, the oncogenic Ha-ras G12V gene, or mini-gene constructs of wild-type Ki-ras4B as a control. Ki-rasmutations, but not Ha-ras mutations, disrupted colon epithelial cell apicobasal polarity and adhesion to collagen I and laminin. Three Ha-ras transfectants and three Ki-ras transfectants exhibited Ras proteins expressing the Val-12 mutation by Western blotting with pan-ras G12V antibody. Only wild-type Ki-ras transfectant cells and oncogenic Ha-rastransfectant cells synthesized the mature, fully glycosylated forms of β1 integrin. Instead of the mature integrin β1-chain, a faster migrating β1-chain intermediate was detected on the cell surface and in the cytoplasm of the oncogenic Ki-ras transfectants. Expression of the oncogenic Ki-ras gene caused the altered β1 integrin maturation because phosphorothiolated antisense oligonucleotides to Ki-ras reduced expression of both the mutant Ki-Ras protein and the aberrant integrin β1-chain and increased expression of the mature integrin β1-chain. Altered glycosylation generated the new β1 integrin form since integrin core β1-chain proteins of the same molecular weight were yielded in Ki-ras, Ha-ras, and control transfectants after removal of sugar residues with endoglycosidase F or following tunicamycin treatment to inhibit glycosylation. The selective effect of oncogenic Ki-ras on β1 integrin glycosylation was not due to selective activation of mitogen-activated protein kinases because both mutated Ki- and Ha-ras genes activated this pathway and increased cell proliferation. Since blocking the glycosylation of integrin β1-chain inhibited the adherence, polarization, and subsequent differentiation of colon epithelial cells, the selective effects of the oncogenic cellular Ki-ras gene on integrin β1-chain glycosylation may account, at least in part, for the selection of Ki-ras mutations in human colon tumors.

Igfbp-3 mediates TGFβ1 proliferative response in colon cancer cells

Many human tumor cells are resistant to growth inhibition by TGFβ1. Resistance may be caused by mutations in TGFβ receptors or in other components of the TGFβ signal transduction systems, or by knockout of the retinoblastoma (Rb) gene, which in fibroblasts converts cellular response to TGFβ1 from growth inhibition to growth stimulation. Our earlier studies showed such a switch in response to TGFβ1 occurred in 45% of colon cancers but without deletion of Rb. We now show that insulin‐like growth factor binding protein 3 (IGFBP‐3) mediates the TGFβ1‐induced proliferation of 3 metastatic or highly aggressive colon carcinoma cell lines. TGFβ1 increases IGFBP‐3 abundance while phosphorothiolated antisense oligonucleotides to IGFBP‐3 block the growth‐promoting effect of TGFβ1 in each of 3 lines.IGFBP‐3 induces carcinoma cell growth in a dose‐dependent and time‐dependent manner in vitro. IGFBP‐3 may confer a selective growth advantage on tumor cells in vivo because levels of mature IGFBP‐3 were elevated at least 2‐fold in 7 of 10 resected colon cancers compared with adjacent normal tissue. Int. J. Cancer 87:373–378, 2000.

Oncogenic c-Ki-ras but Not Oncogenic c-Ha-ras Up-regulates CEA Expression and Disrupts Basolateral Polarity in Colon Epithelial Cells

Colon carcinomas commonly contain mutations in Ki-ras4B, but very rarely in Ha-ras, suggesting that different Ras isoforms may have distinct functions in colon epithelial cell biology. In an earlier study we had demonstrated that oncogenic Ki-ras4BVal-12, but not oncogenic Ha-ras Val-12, blocks the apicobasal polarization of colon epithelial cells by preventing normal glycosylation of the integrin β1 chain of the collagen receptor. As a result, only the Ki-ras mutated cells exhibited altered cell to substratum attachment, whereas mutation of either Ras isoform activated mitogen-activated protein kinases. We have now asked whether intercellular adhesion proteins implicated in establishing basolateral polarity in colon epithelial cells are modulated by oncogenic Ki-Ras4BVal-12 proteins but not oncogenic Ha-RasVal-12 proteins. The embryonic adhesion protein carcinoembryonic antigen (CEA) was up-regulated on the mRNA and protein levels in each of three stable Ki-ras Val-12 transfectant lines but in none of three stable Ha-ras Val-12 transfectant lines. The elevated protein levels of CEA in Ki-ras4BVal-12 transfectant cells were decreased by blocking expression of Ki-ras4BVal-12 with antisense oligonucleotides. N-cadherin levels were decreased in only the Ki-rastransfectants, whereas E-cadherin levels were unchanged. Immunohistochemical analysis demonstrated that Ki-ras4BVal-12 transfectant cells did not polarize into cells with discrete apical and basal regions and so could not restrict expression of CEA to the apical region. These unpolarized cells displayed elevated levels of CEA all along their surface membrane where CEA mediated random, multilayered associations of tumor cells. This aggregation was both calcium-independent and blocked by Fab′ fragments of anti-CEA monoclonal antibody col-1. Trafficking of the lysosomal cysteine protease cathepsin B may also be altered when cell polarity cannot be established. Ki-ras4BVal-12transfectant cells expressed 2-fold elevated protein levels of the lysosomal cysteine protease cathepsin B but did not up-regulate cathepsin B mRNA expression. One function of oncogenic c-Ki-Ras proteins in colon cancer progression may be to up-regulate CEA and thus to prevent the lateral adhesion of adjacent colon epithelial cells that normally form a monolayer in vivo.

Transforming Growth Factor β1 Induces Proliferation in Colon Carcinoma Cells by Ras-dependent, smad-independent Down-regulation of p21cip1

Transforming growth factor β1 (TGFβ1) can act as a tumor suppressor or a tumor promoter depending on the characteristics of the malignant cell. We recently demonstrated that colon carcinoma cells transfected with oncogenic cellularK-rasV12, but not oncogenic cellular H-rasV12, switched from TGFβ1-insensitive to TGFβ1-growth-stimulated and also became more invasive (Yan, Z., Deng, X., and Friedman, E. (2001)J. Biol. Chem. 276, 1555–1563). We now demonstrate that TGFβ1 growth stimulation of colon carcinoma cells is Ras-dependent and smad-independent. In U9 colon carcinoma cells, which are responsive to TGFβ1 by growth stimulation, a truncating mutation at Gln-311 was found in thesmad4 gene. Very little smad4 protein was detected in these cells. Loss of smad4 protein was confirmed by functional studies. In U9 cells co-transfected wild-type smad4, but not mutant smad4, mediated response of the 3TP-lux and pSBE promoter reporter constructs to TGFβ1. Proliferation initiated by TGFβ1 in U9 cells required Ras-mediated down-regulation of p21cip1 protein. Less p21cip1 was associated with cdk2·cyclin complexes in TGFβ1-treated U9 cells, and the cdk2 complexes had increased kinase activity. Elevation of p21cip1 levels diminished proliferative response to TGFβ1. U9 cells expressing DN-N17ras neither proliferated in response to TGFβ1 nor down-regulated the cdk inhibitor p21cip1, and TGFβ1 activation of 3TP-lux in U9 cells was inhibited by DN-N17ras in a dose-dependent manner. TGFβ1 also decreased p21cip1 levels and stimulated proliferation in SW480 cells, which express mutant K-Ras but no smad4 protein. TGFβ1 did not activate or inhibit the p21cip1 promoter construct in U9 cells even in the presence of co-transfected smad4, or alter p21cip1 mRNA levels. Thus the decrease in p21cip1 levels was mediated by a TGFβ-initiated Ras-dependent, but smad-independent post-transcriptional mechanism.

Short-chain fatty acids induce cell cycle inhibitors in colonocytes.

BACKGROUND & AIMSnWe tested the hypothesis that short-chain organic acids in the colon derived from dietary pectin, wheat bran, and oat bran are protective against the development of colon cancer because they induce transforming growth factor (TGF)-beta1, which in turn inhibits cell growth by inducing cyclin-directed kinase (cdk) inhibitors.nnnMETHODSnU4 human colon carcinoma cells differentiate into water- and salt-transporting columnar enterocytes and therefore model normal colonocytes. The composition and kinase activity of cdk/cyclin complexes were determined by immunoprecipitation and Western blotting studies in U4 cells treated in vitro with short-chain fatty acid (SCFA) mixtures that mimic the digestion products of wheat bran, oat bran, pectin, and cellulose (as control), which is largely unfermentable.nnnRESULTSnInduction of the cdk inhibitors p21cip1 and p27kip1 by fiber-mimicking SCFA mixtures occurs much more rapidly and is many-fold greater than their induction by TGF-beta1. The SCFA mixtures most effective in causing growth inhibition and cdk inhibitor production mimicked those from wheat bran > oat bran > pectin.nnnCONCLUSIONSncdk inhibitor induction by SCFA mixtures is not mediated by TGF-beta1. The SCFA mixture mimicking digested wheat bran fiber was the most effective of all mixtures tested in inhibiting cell growth through induction of cdk inhibitors.

Rapid turnover of cell-cycle regulators found in Mirk/dyrk1B transfectants.

Mirk/dyrk1B is an arginine‐directed protein kinase, which functions as a transcriptional activator and mediates serum‐free growth of colon carcinoma cells by an unknown mechanism. We now report that turnover of the cdk inhibitor p27kip1 and the G1‐phase cyclin cyclin D1 is enhanced in each of 4 Mirk stable transfectants compared to vector control transfectants and Mirk kinase‐inactive mutant transfectants. This enhanced turnover is proteasome‐dependent and leads to lower protein levels of both p27kip1 and cyclin D1. Lower protein levels of the cdk inhibitor p21cip1 were also observed in the 4 Mirk stable transfectants. Mirk did not alter the activity of a p27kip1 promoter construct or p27kip1 mRNA levels by stable expression, indicating that the decrease in p27kip1 protein levels was due to a posttranscriptional mechanism. These data are consistent with mirk enhancing the expression of some component common to the proteolysis of both p27kip1 and cyclin D1.

Insulin‐like growth factor‐I has a biphasic effect on colon carcinoma cells through transient inactivation of forkhead1, initially mitogenic, then mediating growth arrest and differentiation

IGF‐I stimulates intestinal cell differentiation after initiating a short proliferative burst, similar to its effect on muscle cell differentiation. Levels of IGF‐I attainable in serum (10–20 ng/ml) induced transient growth stimulation of colon carcinoma cells, then growth arrest. When IGF‐I functioned as a mitogen, it blocked differentiation. Intestinal cell differentiation occurred once cells had undergone the IGF‐I–initiated growth arrest and IGF‐I and butyrate acted synergistically to induce maturation markers. IGF‐I induces NIH‐3T3 cell proliferation and survival by activating the kinase akt, which in turn inhibits various apoptotic mediators and the forkhead family of transcription factors, which mediate expression of p27kip1. Promoter reporter assays demonstrated that forkhead1 mediates transcription of p27kip1 in colon carcinoma cells. The mitogenic effects of IGF‐I on 4 colon carcinoma cell lines were transient because the inactivating phosphorylation of forkhead1 by akt was short‐lived. This allowed transcriptional upregulation of the cdk inhibitor p27kip1, with a resulting growth arrest. In contrast, in NIH‐3T3 cells treated in parallel with identical IGF‐I levels, forkhead phosphorylation levels were sustained; thus, no increase in p27kip1 levels was seen and cells continued to proliferate. Intestinal epithelial cells in vivo undergo a limited number of divisions, then growth arrest and completion of their maturation. IGFs found in intestinal tissue may control the timing of this process. In addition, colon cancers may have developed strategies to overcome IGF‐I–mediated growth arrest. Earlier (Kansra et al., Int J Cancer 2000;87:373–8), we found that levels of IGFBP‐3 were elevated at least 2‐fold in 70% of resected colon cancers compared with adjacent normal tissue. In the current study, growth inhibition by IGF‐I and IGF‐II was blocked by concurrent addition of IGFBP‐3, implying that colon cancers with elevated IGFBP‐3 levels would be selected for in vivo because they could bind and inactivate high serum IGF‐I levels and continue to proliferate.

Transient arrest in a quiescent state allows ovarian cancer cells to survive suboptimal growth conditions and is mediated by both Mirk/dyrk1b and p130/Rb2

Some ovarian cancer cells in vivo are in a reversible quiescent state where they can contribute to cancer spread under favorable growth conditions. The serine/threonine kinase Mirk/dyrk1B was expressed in each of seven ovarian cancer cell lines and in 21 of 28 resected human ovarian cancers, and upregulated in 60% of the cancers. Some ovarian cancer cells were found in a G0 quiescent state, with the highest fraction in a line with an amplified Mirk gene. Suboptimal culture conditions increased the G0 fraction in SKOV3 and TOV21G, but not OVCAR4 cultures. Less than half as many OVCAR4 cells survived under suboptimal culture conditions as shown by total cell numbers, dye exclusion viability studies, and assay of cleaved apoptotic marker proteins. G0 arrest in TOV21G and SKOV3 cells led to increased levels of Mirk, the CDK inhibitor p27, p130/Rb2, and p130/Rb2 complexed with E2F4. The G0 arrest was transient, and cells exited G0 when fresh nutrients were supplied. Depletion of p130/Rb2 reduced the G0 fraction, increased cell sensitivity to serum‐free culture and to cisplatin, and reduced Mirk levels. Mirk contributed to G0 arrest by destabilization of cyclin D1. In TOV21G cells, but not in normal diploid fibroblasts, Mirk depletion led to increased apoptosis and loss of viability. Because Mirk is expressed at low levels in most normal adult tissues, the elevated Mirk protein levels in ovarian cancers may present a novel therapeutic target, in particular for quiescent tumor cells which are difficult to eradicate by conventional therapies targeting dividing cells.

Downregulation of p53 by sustained JNK activation during apoptosis.

In a previous study, we prepared short‐chain fatty acid (SCFA) mixtures mimicking the composition of the digested fibers from wheat bran, oat bran, pectin, and cellulose and tested the products on U4 cells, a cell‐line model for normal colonocytes. These SCFA mixes induced the cyclin‐dependent kinase (cdk) inhibitors p21 and p27, which bound to cdk2/cyclin E and cdk4/cyclin D1 complexes, blocking their kinase activity and arresting cell growth. SCFAs from digested fiber may control intestinal crypt height in vivo by inducing apoptosis in growth‐arrested cells at the top of the crypt. In the present study, we report that SCFA mixes induced apoptosis of U4 cells and unexpectedly caused both a sustained activation of the stress‐activated protein kinase c‐jun N‐terminal kinase 1 (JNK1) and downregulation of the tumor suppressor protein p53. JNK1 bound to p53, and the amount of JNK1‐bound p53 accurately reflected the amount of total cellular p53. After activation by SCFAs, JNK1 phosphorylated its bound p53. This phosphorylation is likely to have converted p53 into an apoptotic target because p53 breakdown correlated with caspase‐3 activity, was inhibited by a caspase‐3 inhibitor in a dose‐dependent manner, and was inhibited by transfection of dominant‐negative JNK1. Because JNK1 activation was sustained in SCFA‐treated U4 cells, JNK1 can bind, phosphorylate, and release p53 for proteolysis and then continue this cycle until many p53 molecules have been phosphorylated. Loss of p53 protein was likely due to proteolysis and not to transcriptional changes because a sixfold decrease in p53 protein occurred within 3–24u2009h of SCFA treatment, whereas p53 mRNA levels were downregulated as much only after 2–3u2009d. SCFA mixes targeted p53 and possibly other cellular proteins for degradation during apoptosis by causing a sustained activation of JNKs. Mol. Carcinog. 29:179–188, 2000.