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. Author manuscript; available in PMC: 2011 Jan 20.
Published in final edited form as: Cancer Biol Ther. 2008 Dec 9;7(12):1959–1967. doi: 10.4161/cbt.7.12.6956

MUC1 oncoprotein suppresses activation of the ARF-MDM2-p53 pathway

Deepak Raina 1, Rehan Ahmad 1, Dongshu Chen 1,†, Shailendra Kumar 1,‡, Surender Kharbanda 1, Donald Kufe 1,*
PMCID: PMC3023907  NIHMSID: NIHMS263636  PMID: 18981727

Abstract

The MUC1 oncoprotein interacts with the c-Abl tyrosine kinase and blocks nuclear targeting of c-Abl in the apoptotic response to DNA damage. Mutation of the MUC1 cytoplasmic domain at Tyr-60 disrupts the MUC1-c-Abl interaction. The present results demonstrate that the MUC1(Y60F) mutant is a potent inducer of the ARF tumor suppressor. MUC1(Y60F) induces transcription of the ARF locus by a c-Abl-dependent mechanism that promotes CUL-4A-mediated nuclear export of the replication protein Cdc6. The functional significance of these findings is that MUC1(Y60F)-induced ARF expression and thereby inhibition of MDM2 results in the upregulation of p53 and the homeodomain interacting protein kinase 2 (HIPK2) serine/threonine kinase. HIPK2-mediated phosphorylation of p53 on Ser-46 was further associated with a shift from expression of the cell cycle arrest-related p21 gene to the apoptosis-related PUMA gene. We also show that the MUC1(Y60F) mutant functions as dominant negative inhibitor of tumorigenicity. These findings indicate that the oncogenic function of MUC1 is conferred by suppressing activation of the ARF-MDM2-p53 pathway.

Keywords: MUC1, ARF, MDM2, p53, HIPK2, INK4

Introduction

The heterodimeric MUC1 protein is aberrantly overexpressed by diverse human carcinomas and certain hematologic malignancies. MUC1 is positioned at the cell membrane as a complex of N- and C-terminal subunits. The MUC1 N-terminal subunit (MUC1-N) largely consists of glycosylated 20 amino acid tandem repeats and extends beyond the glycocalyx of the cell.1,2 MUC1-N forms a stable complex with the transmembrane MUC1 C-terminal subunit (MUC1-C) that includes a 58 amino acid extracellular domain and a 72 amino acid cytoplasmic domain.3 At the cell membrane, MUC1-C interacts with receptor tyrosine kinases in part through the extracellular domain and a bridge mediated by galectin-3.4–8 MUC1-C is also targeted to (i) the mitochondrial outer membrane by HSP70 and HSP90,6,9,10 and (ii) the nucleus by importin β.11,12 The MUC1-C cytoplasmic domain is phosphorylated by multiple kinases13–16 and associates with certain transcription factors.17–19 The available evidence indicates that MUC1-C directly regulates the Wnt/β-catenin,13,20,21 p53,17,19 and NFκB22 pathways that have been linked to transformation. MUC1-C also blocks the apoptotic response to stress9,17,21,23–26 and induces transformation.11,21,27 Moreover, transgenic mouse models have demonstrated that MUC1 contributes to tumor development.28,29

The p53 tumor suppressor functions in the cellular response to stress by inducing growth arrest, DNA repair, apoptosis or senescence. 30 Direct binding of MDM2 to the p53 N-terminus inhibits the p53 transactivation function.31,32 The E3 ubiquitin ligase activity of MDM2 also promotes the ubiquitination and proteosomal degradation of p53.33,34 The MDM2 gene is transcriptionally regulated by p53, thus establishing a p53-MDM2 feedback loop. Conversely, the p19ARF (ARF) tumor suppressor stabilizes and activates p53 by directly inhibiting MDM2.35 The INK4b-ARF-INK4a locus on human chromosome 9 encodes ARF and the p15INK4b/p16INK4a inhibitors of the G1 cyclin-dependent kinases.35,36 Transcription of the INK4b-ARF-INK4a locus is repressed by binding of the Cdc6 regulatory protein to a cis-acting DNA replication origin.37 Other studies have shown that c-Abl activates the CUL-4A ubiquitin ligase38 and that CUL-4 promotes the nuclear export of Cdc6 by negative regulation of the cyclin-dependent kinase inhibitor, CKI-1.39 The INK4b, ARF and INK4a genes have also been shown to be independently regulated by diverse signaling pathways.36 For example, ARF is not expressed in most normal tissues; however, overexpression or mutational activation of oncoproteins activates transcription of ARF and not the INK4b or INK4a genes.36

The present studies demonstrate that, unlike certain other oncoproteins, MUC1 is not associated with upregulation of ARF expression. In this regard, we show that mutation of the MUC1-C cytoplasmic domain at Tyr-60 activates ARF gene transcription and stabilization of p53 and the HIPK2 kinase. MUC1(Y60F) induces ARF expression by a mechanism involving c-Abl, CUL-4A and Cdc6. Consistent with upregulation of ARF, MUC1(Y60F) functions as a dominant negative of the malignant phenotype. These findings indicate that MUC1 suppresses activation of the ARF-MDM2-p53 pathway.

Results

MUC1(Y60F) mutant confers stabilization of p53

HCT116 cells stably expressing an empty vector, MUC1 or MUC1(Y60F) were analyzed for p53 levels. As compared to that in HCT116/vector cells, stable expression of wild-type MUC1 had little effect on p53 levels (Fig. 1A). By contrast, p53 expression was found to be substantially higher in HCT116/MUC1(Y60F) cells (Fig. 1A). Similar results were obtained in separately isolated HCT116 cell clones expressing MUC1 or MUC1(Y60F) (Fig. 1A). Subcellular fractionation further demonstrated that MUC1(Y60F) increases p53 expression in the nucleus (Fig. 1B). Treatment of the HCT116 cells with CHX to inhibit protein synthesis was associated with a rapid decrease in p53 levels in HCT116/vector and HCT116/MUC1 cells, consistent with the short half-life of the p53 protein (Fig. 1C). Notably, however, CHX had no apparent effect on p53 levels in HCT116/MUC1(Y60F) cells (Fig. 1C). In addition, treatment with CHX in the presence of MG132 to inhibit the proteosome stabilized p53 in the HCT116/vector and HCT116/MUC1 cells, but had little effect on the constitutively high p53 levels in HCT116/MUC1(Y60F) cells (Fig. 1D). These results indicated that the MUC1(Y60F) mutant increases p53 expression by blocking p53 degradation.

Figure 1.

Figure 1

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MUC1(Y60F) increases p53 expression by stabilizing p53. (A) Whole cell lysates from HCT116 cells stably expressing the empty vector, MUC1 or MUC1(Y60F) were immunoblotted with the indicated antibodies. (A and B) denotes two separately isolated clones. (B) Nuclear lysates from the HCT116/vector, HCT116/MUC1 and HCT116/MUC1(Y60F) cells were immunoblotted with antibodies against p53 and nuclear lamin B. (C) The indicated HCT116 cells were treated with CHX for 0–8 hours. Lysates were immunoblotted with anti-p53 and anti-β-actin (left). Intensity of the p53 signals was determined by densitometric scanning and is presented as % of control levels over time of CHX treatment for HCT116/vector (□), HCT116MUC1 (■) and HCT116/MUC1(Y60F) (●) cells (right). (D) The indicated HCT116 cells were treated with MG132 and CHX for 0–8 hours. Lysates were immunoblotted with the indicated antibodies.

MUC1(Y60F) regulates HIPK2 levels

HIPK2 is a serine/threonine kinase that activates p53 and, like p53, is a target for MDM2-mediated degradation.40–42 Compared to HCT116/vector cells, HIPK2 levels were decreased by wild-type MUC1 and increased by the MUC1(Y60F) mutant (Fig. 2A, left). These results were confirmed by densitometric scanning of the signals from different immunoblots (Fig. 2A, right). Stability of HIPK2 in the presence of CHX was also increased in HCT116/MUC1(Y60F) cells as compared to HCT116/vector and HCT116/MUC1 cells (Fig. 2B, left). Thus, the half-life of HIPK2 was 6 h and >8 h in HCT116/MUC1 and HCT116/vector cells, respectively (Fig. 4B, right). In addition, whereas MG132 blocked the CHX-induced decreases in HIPK2 levels in HCT116/vector and HCT116/MUC1 cells, inhibition of the proteosome had little effect on HIPK2 levels in HCT116/MUC1(Y60F) cells (Fig. 2C). The MUC1 cytoplasmic domain regulates c-Abl by a mechanism dependent on the Tyr-60 site.16 To determine whether c-Abl contributes to the regulation of HIPK2, we silenced c-Abl in the HCT116/MUC1(Y60F) cells by transfection with a c-AblsiRNA (Fig. 2D). Notably, silencing of c-Abl was associated with downregulation of HIPK2 (Fig. 2D). These findings indicate that HIPK2 levels are regulated by MUC1 and c-Abl.

Figure 2.

Figure 2

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MUC1 regulates stability of HIPK2. (A) Lysates from the indicated HCT116 cells were immunoblotted with anti-HIPK2 and anti-β-actin (left). Densitometric scanning of the signal intensities was obtained from two independent immunoblots. The results are expressed as the relative HIPK2 level (mean ± SD) compared to that in HCT116/vector cells (assigned a value of 1) (right). (B) The indicated HCT116 cells were treated with CHX for 0–8 hours. Lysates were immunoblotted with anti-HIPK2 and anti-β-actin (left). Intensity of the p53 signals was determined by densitometric scanning and is presented as % of control levels over time of CHX treatment for HCT116/vector (□), HCT116MUC1 (■) and HCT116/MUC1(Y60F) (●) cells (right). (C) The indicated HCT116 cells were treated with MG132 and CHX for 0–8 hours. Lysates were immunoblotted with the indicated antibodies. (D) HCT116/MUC1(Y60F) cells were transfected with control CsiRNA or c-AblsiRNA pools. At 72 hours after transfection, lysates were immunoblotted with the indicated antibodies.

Figure 4.

Figure 4

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MUC1(Y60F) regulates nuclear localization of Cdc6 by a c-Abl-dependent mechanism. (A) Whole cell lysates from the indicated HCT116 cells were immunoblotted with anti-Cdc6 and anti-β-actin. (B) Nuclear lysates from the indicated cells were immunoblotted with anti-Cdc6 and anti-lamin B. (C) Soluble chromatin from the indicated cells was immunoprecipitated with anti-Cdc6. The precipitates were analyzed by PCR for RD (upper) and control region (CR; lower) ARF promoter sequences. (D) HCT116/MUC1(Y60F) cells were transfected with control CsiRNA or c-AblsiRNA pools. Whole cell lysates (left) and nuclear lysates (right) were immunoblotted with the indicated antibodies.

MUC1(Y60F) mutant induces ARF expression

The findings that MUC1(Y60F) increases p53 and HIPK2 levels supported the potential involvement of MDM2, which contributes to the degradation of both proteins. There was, however, no detectable effect of MUC1 or MUC1(Y60F) on MDM2 levels (Fig. 3A). By contrast, expression of the MDM2 inhibitor, ARF, was markedly upregulated in the HCT116/MUC1(Y60F) cells (Fig. 3A). The INK4/ARF locus encodes ARF and the INK4a and INK4b tumor suppressor proteins.43 ARF and INK4a share common exons 2 and 3 with the two proteins encoded by alternate reading frames. As found for ARF, INK4a expression was also upregulated in HCT116/MUC1(Y60F) cells (Fig. 3A). In addition, INK4b expression was constitutively upregulated in HCT116/vector cells and there was little if any effect of MUC1 or MUC1(Y60F) on INK4b levels (Fig. 3A). Transfection of the HCT116 cells with an ARF promoter ligated upstream to luciferase (pARF-Luc) demonstrated that the ARF promoter is activated about five fold by MUC1(Y60F) as compared to that in the absence or presence of wild-type MUC1 (Fig. 3B). In concert with these results, ARF mRNA levels were also increased in the HCT116/MUC1(Y60F) cells (Fig. 3C), indicating that the Y60F mutation is associated with transcriptional activation of the ARF locus.

Figure 3.

Figure 3

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MUC1(Y60F) activates the ARF locus. (A) Lysates from the HCT116/vector, HCT116/MUC1 and HCT116/MUC1(Y60F) cells were immunoblotted with the indicated antibodies. (B) The indicated HCT116 cells were transfected with pARF-Luc and SV-40-Renilla-Luc. Luciferase activity was measured at 48 hours after transfection. The results are expressed as the fold-activation (mean ± SD of three independent experiments) compared with that obtained in HCT116/MUC1 cells (assigned a value of 1). (C) ARF and β-actin mRNA levels were determined for the indicated cells by quantitative RT-PCR.

MUC1(Y60F) regulates nuclear localization of Cdc6 by a c-Abl- and CUL-4A-dependent mechanism

The replication protein Cdc6 represses transcription of the INK4/ARF locus.44 Expression of wild-type MUC1 and the MUC1(Y60F) mutant was associated with increases in Cdc6 levels (Fig. 4A). However, in contrast to wild-type MUC1, nuclear levels of Cdc6 were decreased with the MUC1(Y60F) mutant (Fig. 4B). In concert with these findings, Cdc6 occupancy of the ARF promoter was detectable in cells expressing wild-type MUC1 and not the MUC1(Y60F) mutant (Fig. 4C). Whereas MUC1 and not MUC1(Y60F) interacts with c-Abl in the cytoplasm (Raina et al., 2006), we transiently silenced c-Abl in HCT116/MUC1(Y60F) cells (Fig. 4D, left). Downregulation of c-Abl had little effect on Cdc6 and MUC1-C levels (Fig. 4C), but substantially increased targeting of Cdc6 to the nucleus (Fig. 4D, right).

c-Abl promotes activation of CUL-4A and thereby nuclear export of Cdc6.38,39 Silencing of CUL-4A had no apparent effect on c-Abl or Cdc6 levels (Fig. 5A). However, silencing of CUL-4A was associated with nuclear targeting of Cdc6 (Fig. 5B). Moreover, silencing of CUL-4A was associated with downregulation of ARF, INK4a and INK4b (Fig. 5C). In concert with c-Abl-mediated activation of CUL-4A, downregulation of c-Abl had similar effects on ARF and INK4a expression (Fig. 5D). Silencing of a c-Abl was also associated with a partial decrease in INK4b levels (Fig. 5D), consistent with regulation of INK4b gene by both c-Abl-dependent and -independent mechanisms. These results collectively indicate that the MUC1(Y60F) mutant induces ARF expression by a mechanism involving dysregulation of c-Abl, CUL-4A and Cdc6.

Figure 5.

Figure 5

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MUC1(Y60F) regulates nuclear localization of Cdc6 by a CUL-4A-dependent mechanism. (A–C) HCT116/MUC1(Y60F) cells were transfected with control CsiRNA or CUL-4AsiRNA pools. Whole cell lysates (A and C) and nuclear lysates (B) were immunoblotted with the indicated antibodies. (D) HCT116/MUC1(Y60F) cells were transfected with control CsiRNA or c-AblsiRNA pools. Whole cell lysates were immunoblotted with the indicated antibodies.

MUC1(Y60F) induces phosphorylation of p53 on Ser-46

HIPK2 phosphorylates p53 on Ser-46.45 Thus, a potential outcome of the upregulation of both p53 and HIPK2 by MUC1(Y60F) is an increase in p53 phosphorylation. In this regard, phosphorylation of p53 on Ser-46 was detectable constitutively in HCT116/MUC1(Y60F), but not in HCT116/vector or HCT116/MUC1, cells (Fig. 6A). Moreover, phosphorylation of p53 on Ser-46 was higher in the response of HCT116/MUC1(Y60F) cells to DNA damage induced by cisplatin (CDDP) treatment (Fig. 6A). Similar results were obtained when the cells were treated with etoposide (Fig. 6B). As shown previously,17 MUC1 promotes p53-mediated induction of p21 (Fig. 6C, left). Notably, however, the induction of p21 expression was abrogated by MUC1(Y60F) (Fig. 6C, left). Activation of a p21-luciferase reporter further demonstrated that, compared to MUC1, the p21 promoter is downregulated by MUC1(Y60F) (Fig. 6C, right). By contrast and consistent with the phosphorylation of p53 on Ser-46, CDDP-induced expression of PUMA was attenuated by MUC1 and increased by MUC1(Y60F) (Fig. 6D, left). Studies with a PUMA-luciferase reporter confirmed that CDDP-induced activation of the PUMA promoter is suppressed by MUC1 and stimulated by MUC1(Y60F) (Fig. 6D, right). These results indicate that expression of the MUC1(Y60F) mutant increases phosphorylation of p53 on Ser-46 and thereby promotes a shift from p21 to PUMA expression.

Figure 6.

Figure 6

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MUC1(Y60F) induces phosphorylation of p53 on Ser-46. (A and B) The indicated HCT116 cells were treated with CDDP (A) or etoposide (B) for 0, 6 and 12 hours. Lysates were immunoblotted with the indicated antibodies. (C) HCT116/vector, HCT116/MUC1 and HCT116/MUC1(Y60F) cells were treated with CDDP for the indicated times (left). Lysates were immunoblotted with anti-p21 and anti-β-actin (left). The indicated HCT116 cells were transfected with p21-Luc and SV40-Renilla-Luc. Luciferase activity was measured at 48 hours after transfection. The results are expressed as fold-activation (mean ± SD of three independent experiments) compared to that obtained with HCT116/MUC1(Y60F) cells (assigned a value of 1) (right). (D) HCT116/vector, HCT116/MUC1 and HCT116/MUC1(Y60F) cells were treated with CDDP for the indicated times. Lysates were immunoblotted with anti-PUMA and anti-β-actin (left). The indicated HCT116 cells were transfected with pPUMA-Luc and SV40-Renilla-Luc (right). At 24 hours after transfection, the cells were left untreated (open bars) or treated with CDDP (solid bars) for 24 hours and then assayed for luciferase activity. The results are expressed as fold-activation (mean ± SD) for three independent experiments) compared to that obtained with untreated HCT116/vector cells (assigned a value of 1) (right).

MUC1(Y60F) functions as a dominant negative for transformation

To determine whether MUC1(Y60F) affects the MUC1 transforming function, the HCT116 cell transfectants were assessed for anchorage-dependent and -independent growth. Expression of MUC1 or MUC1(Y60F) had no effect on anchorage-dependent growth (data not shown). However, consistent with previous findings,15,27 HCT116/MUC1 cells formed colonies that were substantially larger than those found with HCT116/vector cells (Fig. 7A). In contrast, expression of MUC1(Y60F) was associated with the formation of colonies that were similar to those obtained with HCT116/vector cells (Fig. 7A). The plating efficiency was ~15% for HCT116/MUC1 cells and less than 0.1% for HCT116/vector and HCT116/MUC1(Y60F) cells (Fig. 7B). To determine whether expression of MUC1(Y60F) affects tumorigenicity, the HCT116 transfectants were injected subcutaneously in nude mice. As found previously,15,27 tumors formed with HCT116/MUC1 cells were larger than those obtained with HCT116/vector cells (Fig. 7C). Notably, however, HCT116/MUC1(Y60F) cells formed tumors that were substantially smaller than those in mice injected with HCT116/MUC1 and HCT116/vector cells (Fig. 7C). These findings indicate that the MUC1(Y60F) mutant is not functional in supporting anchorage-independent growth and attenuates tumorigenicity of HCT116 cells.

Figure 7.

Figure 7

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MUC1(Y60F) functions as a dominant-negative for tumorigenicity. (A) The indicated cells were suspended in soft agar and incubated for three weeks. Representative photomicrographs depict colony formation. (B) Colonies larger than 10 cells were counted for each of the indicated cells. The results are expressed as the number of colonies (mean ± SD for three independent experiments). (C) The indicated cells were injected subcutaneously into the posterior flanks of nude mice. Tumor volumes were calculated from bidimensional measurements on day 30. The results are expressed as the tumor volume (mean ± SD) obtained from six mice/group. (D) Schema depicting the proposed interactions by which MUC1-C regulates the c-Abl→CUL-4A→Cdc6 pathway. Wild-type MUC1-C interacts with c-Abl and thereby prevents c-Abl from activating CUL-4A (upper). In turn, Cdc6 suppresses transcription of the ARF locus (upper). The MUC1(Y60F) mutation abrogates the interaction with c-Abl, which permits c-Abl activation of CUL-4A and release of Cdc6-mediated transcriptional suppression (lower).

Discussion

MUC1 regulates activation of the ARF locus by a c-Abl-dependent mechanism

Shuttling of c-Abl between the cytoplasm and nucleus is in part regulated through interactions with 14-3-3 proteins.46 Direct binding of the c-Abl SH2 domain to the pTyr-60 site in the MUC1 cytoplasmic domain disrupts the c-Abl-14-3-3 interaction and sequesters c-Abl in the cytoplasm.16 In this regard, previous work showed that mutation of Tyr-60 attenuates (i) binding of MUC1 and c-Abl, (ii) sequestration of c-Abl in the cytoplasm and (iii) the MUC1 antiapoptotic function. The present studies demonstrate that the interaction between MUC1 and c-Abl is involved in regulating expression of the ARF tumor suppressor. Thus, expression of MUC1 with the Y60F mutation was associated with a substantial upregulation of ARF gene transcription. Moreover, silencing c-Abl abrogated the induction of ARF expression by the MUC1(Y60F) mutant. The results also demonstrate that mutating MUC1 at Tyr-60 upregulates INK4a expression, but not INK4b, which is expressed constitutively in HCT116 cells. Upregulation of INK4a was similarly blocked by silencing c-Abl, indicating that MUC1(Y60F) activates the ARF and INK4a genes by a c-Abl-dependent mechanism. Conversely, wild-type MUC1 sequesters c-Abl in the cytoplasm and thereby suppresses the function of c-Abl in inducing ARF and INK4a expression (Fig. 7D).

MUC1(Y60F) induces ARF through CUL-4A

The CUL-4A ubiquitin ligase promotes proteosomal degradation of damage-specific DNA binding proteins (DDBs).47 c-Abl forms complexes with DDBs48 and was recently found to stimulate CUL-4A activity.38 CUL-4 also prevents DNA rereplication by blocking assembly of prereplicative complexes that contain the replication-licensing factors Cdc6 and Cdt1. In this context, CUL-4A targets Cdt1 for degradation and promotes the nuclear export of Cdc6.39,49–51 Whereas Cdc6 also represses transcription of the INK4/ARF locus,44 CUL-4A-mediated export of Cdc6 from the nucleus could be associated with activation of ARF expression. The present results demonstrate that both MUC1 and MUC1(Y60F) increase Cdc6 expression; however, nuclear levels of Cdc6 are increased by MUC1 and not MUC1(Y60F). Importantly, silencing of c-Abl or CUL-4A in HCT116/MUC1(Y60F) cells had little effect on Cdc6 levels, but was associated with increased targeting of Cdc6 to the nucleus. In addition, as found for c-Abl, silencing of CUL-4A in the HCT116/MUC1(Y60F) cells was associated with downregulation of ARF and INK4a. Silencing of CUL-4A was also associated with downregulation of INK4b; although, unlike ARF and INK4a, expression of INK4b was decreased only in part by silencing c-Abl. Also, unlike ARF and INK4a, INK4b is constitutively expressed in HCT116 cells, indicating that the INK4b gene is activated by mechanisms independent of MUC1(Y60F) and c-Abl. These findings thus support a model in which MUC1(Y60F) disrupts c-Abl signaling and thereby stimulates CUL-4 activity (Fig. 7D). In turn, CUL-4A promotes nuclear export of Cdc6 and relieves repression of ARF transcription (Fig. 7D).

MUC1(Y60F) increases phosphorylation of p53 on Ser-46

Levels and stability of p53 were substantially increased by MUC1(Y60F), consistent with ARF-mediated inhibition of MDM2. Also consistent with the upregulation of ARF was the finding that MUC1(Y60F) increases expression and stability of HIPK2, another substrate of the MDM2 ubiquitin ligase function that phosphorylates p53 on Ser-46.42 We thus found that upregulation of both p53 and HIPK2 was associated with increased levels of p53 with phosphorylation on Ser-46. The affinity of p53 for different promoters is regulated by Ser-46 phosphorylation with a shift in the p53-mediated cell cycle arrest response to one that induces apoptosis.52–54 Previous work showed that the MUC1 cytoplasmic domain binds directly to the p53 regulatory domain and coactivates p21 gene transcription 17. The present results demonstrate that MUC1(Y60F) is ineffective in coactivating p21 expression. In addition, as found previously for Bax,17 MUC1 attenuated activation of PUMA transcription. Moreover and in contrast, MUC1(Y60F) stimulated the induction of PUMA expression. These findings indicate that, whereas MUC1 promotes the p21 response,17 MUC1(Y60F) confers increased phosphorylation of p53 on Ser-46 and a shift to induction of the proapoptotic PUMA gene.

MUC1(Y60F) blocks tumorigenicity

MUC1, but not MUC1(Y60F), blocks the apoptotic response of HCT116 cells to DNA damage.16 MUC1 also supports growth of HCT116 cells as colonies in agar and as tumors in nude mice.15,27 The present studies demonstrate that mutation of MUC1 at Tyr-60 abrogates the function of MUC1 in supporting anchorage-independent growth. MUC1(Y60F) also inhibited tumorigenicity of HCT116 cells, consistent with a dominant-negative effect. These findings collectively indicate that MUC1 may contribute in part to transformation by blocking c-Abl signaling to the nucleus and the apoptotic response.16 The present findings further indicate that MUC1 functions in preventing activation of a c-Abl→CUL-4A→Cdc6 pathway and induction of ARF transcription. Relief of these MUC1 functions with the Tyr-60 mutation and activation of the ARF gene is thus associated with a block of the malignant HCT116 cell phenotype.

Materials and Methods

Cell culture

Human HCT116/vector, HCT116/MUC1 and HCT116/MUC1(Y60F) cells16 were grown in Dulbecco’s modified Eagle’s medium with 10% heat-inactivated fetal bovine serum (HI-FBS), 100 µg/ml streptomycin, 100 units/ml penicillin and 2 mM L-glutamine. Cells were treated with 10 µg/ml cyclohexamide (CHX; Sigma, St. Louis, MO), 50 µM MG132 (Calbiochem, La Jolla, CA), 30 µM cisplatin (CDDP; Sigma) or 50 µM etoposide (Sigma).

Cell transfections

Cells were seeded (5 × 105/well) on six-well plates. After 24 hours, the cells were transfected with (i) 1 nM SMART c-Abl or nonspecific control siRNA pools (Upstate Cell Signaling Solutions, Charlottesville, VA); or (ii) 1 nM CUL-4A or nonspecific control siRNA pools (Dharmacon/Thermo Scientific, Lafayette, CO) using LipofectAMINE (Invitrogen; Carlsbad, CA). The cells were incubated for an additional 72 hours and then harvested for analysis.

Immunoblot analysis

Whole cell lysates and nuclear fractions prepared from subconfluent cells as described16 were subjected to immunoblot analysis with anti-p53 (Calbiochem), anti-MUC1-C (Ab 5; NeoMarkers, Fremont, CA), anti-β-actin (Sigma), anti-lamin B (Calbiochem), anti-HIPK2 (Abcam, Cambridge, MA), anti-c-Abl (Calbiochem), anti-MDM2 (Calbiochem), anti-ARF (NeoMarkers), anti-INK4a (BD Pharmingen, San Jose, CA), anti-INK4b (Cell Signaling Technology, Danvers, MA), anti-Cdc6 (Thermo Scientific, Fremont, CA), anti-CUL-4A (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-p53-Ser-46 (Cell Signaling Technology), anti-p21 (Santa Cruz Biotechnology) and anti-PUMA (Abcam).

Luciferase assays

Cells were transfected with pARF-Luc, p21-Luc or pPUMA-Luc and SV-40-Renilla-Luc (Promega Life Sciences, Madison, WI) in the presence of LipofectAMINE. After 48 h, the cells were lysed in passive lysis buffer. Lysates were analyzed for firefly and Renilla luciferase activities using the dual luciferase assay kit (Promega).

RT-PCR

Total cellular RNA was extracted using the high pure RNA isolation kit (Roche Diagnostics, Indianapolis, IN). ARF-specific primers (5'-ATC TTG GAG GTC CGG GTG GG-3' and 3'-TGC TGC CCT AGA CGC TGG-5') were designed to amplify a 398-bp fragment. Primers for β-actin were used as a control as described.23 The RNA was reverse-transcribed and amplified using SuperScript One-Step RT-PCR with Platinum Taq (Invitrogen). Amplified fragments were analyzed by electrophoresis in 2% agarose gels.

Chromatin immunoprecipitation (ChIP) assays

Soluble chromatin was immunoprecipitated as described18 using anti-Cdc6 (Ab-1; Thermo Scientific). For PCR, 2 µl from a 50 µl DNA extraction were used with 30–35 cycles of amplification. The primers for the RDINK4/ARF site have been described.37 The primers for the control region (CR) were 5'-CTA ACT GGG GTG AGA TGA TA-3' and 3'-GAT TTT TAA CAA AGA TAT CA-5'.

Anchorage-independent growth

Cells were suspended in 1.5 ml of 0.33% Noble agar (Difco, Detroit, MI) in DMEM supplemented with 10% HI-FBS and antibiotics. The cell suspension was layered over 3.5 ml of 0.5% agar/medium in 60 mm dishes. Colonies composed of >10 cells were counted at three weeks.

Tumorigenicity assays

Cells (1 × 107) were injected subcutaneously in the flanks of 6–8 week old nude mice and tumor volume was determined by bi-dimensional measurements.

Acknowledgements

This work was supported by Grants CA97098 and CA42802 awarded by the National Cancer Institute. The authors thank Gordon Peters, London Research Institute, U.K., for the ARF-luciferase construct and Bert Vogelstein, Johns Hopkins Oncology Center, Baltimore for the PUMA-luciferase vector. The authors acknowledge Kamal Chauhan for technical support.

Abbreviations

MUC1

mucin 1

ChIP

chromatin immunoprecipitation

HIPK2

homeodomain interacting protein kinase 2

CHX

cyclohexamide

CDDP

cisplatin

References

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