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PLK1 is a critical determinant of tumor cell sensitivity to CPT11 and its inhibition enhances the drug antitumor efficacy in squamous cell carcinoma models sensitive and resistant to camptothecins.

Zuco V, De Cesare M, Zaffaroni N, Lanzi C, Cassinelli G - Oncotarget (2015)

Bottom Line: Downregulation of the mitotic kinase PLK1 was found associated with apoptosis induced by SN38 (CPT11 active metabolite).The ability to activate an efficient G2/M cell cycle checkpoint allowing PLK1 ubiquitination and degradation was found associated with SN38-induced apoptosis in SCC cells.A well-tolerated CPT11/BI2536 cotreatment resulted in improved antitumor effect against SCC xenografts in mice compared to single agent treatments.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

ABSTRACT
Intrinsic and acquired tumor drug resistance limits the therapeutic efficacy of camptothecins (CPTs). Downregulation of the mitotic kinase PLK1 was found associated with apoptosis induced by SN38 (CPT11 active metabolite). We investigated the role of PLK1 in the cell response to CPTs in squamous cell carcinoma (SCC) and pediatric sarcoma cell lines and explored the therapeutic potential of the combination of CPT11 and the PLK1 inhibitor BI2536 in CPT-sensitive and -resistant tumor models. Gain- and loss-of-function experiments established a direct role for PLK1 in counteracting SN38 antiproliferative and pro-apoptotic effects. The ability to activate an efficient G2/M cell cycle checkpoint allowing PLK1 ubiquitination and degradation was found associated with SN38-induced apoptosis in SCC cells. However, the synergistic interaction between SN38 and BI2536 enhanced apoptosis in cell lines both sensitive and resistant to SN38-induced apoptotic cell death. A well-tolerated CPT11/BI2536 cotreatment resulted in improved antitumor effect against SCC xenografts in mice compared to single agent treatments. The increased apoptosis induction was reflected in a high rate of complete responses and cures in mice harboring SCC, including tumors with intrinsic or acquired resistance to CPTs. PLK1 inhibition represents a promising strategy to improve the antitumor efficacy of CPT11-based regimens.

No MeSH data available.


Related in: MedlinePlus

Effects of SN38 treatment on PLK1 transcription and ubiquitination and on cell cycle distribution in SCC cell linesCaSki and SiHa cells were treated for 1h with solvent (−) or equimolar concentrations of SN38. A) Twenty four hours later, cells were analyzed for PLK1 mRNA expression by qRT-PCR. B) In parallel, six hours after the end of treatment (1h), cell lysates were subjected to PLK1 immunoprecipitation and analyzed by Western blotting with anti-ubiquitin antibody (in upper panel representative blots are shown). Histograms represent the densitometric quantification of bands corresponding to ubiquitinated relative to not ubiquitinated PLK1 in each cell lines. Data from two independent experiments are expressed in arbitrary units referred to untreated cells (lower panel). C) Cells were exposed to solvent (−) or to the indicated concentrations of SN38 for 1h. Cell lysates were analyzed by Western blotting to evaluate the levels of RPA-2, PLK1, Cdc25A and cell cycle-specific markers. The hyperphosphorylated form of RPA-2 in SiHa cells was evidenced by the electrophoretic mobility shift. Vinculin is shown as a control of protein loading. Lower panels, cell cycle distribution of control and SN38 treated (3μM, for 1h) CaSki and SiHa cells, analyzed 24, 48 and 72 h after treatment.
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Figure 2: Effects of SN38 treatment on PLK1 transcription and ubiquitination and on cell cycle distribution in SCC cell linesCaSki and SiHa cells were treated for 1h with solvent (−) or equimolar concentrations of SN38. A) Twenty four hours later, cells were analyzed for PLK1 mRNA expression by qRT-PCR. B) In parallel, six hours after the end of treatment (1h), cell lysates were subjected to PLK1 immunoprecipitation and analyzed by Western blotting with anti-ubiquitin antibody (in upper panel representative blots are shown). Histograms represent the densitometric quantification of bands corresponding to ubiquitinated relative to not ubiquitinated PLK1 in each cell lines. Data from two independent experiments are expressed in arbitrary units referred to untreated cells (lower panel). C) Cells were exposed to solvent (−) or to the indicated concentrations of SN38 for 1h. Cell lysates were analyzed by Western blotting to evaluate the levels of RPA-2, PLK1, Cdc25A and cell cycle-specific markers. The hyperphosphorylated form of RPA-2 in SiHa cells was evidenced by the electrophoretic mobility shift. Vinculin is shown as a control of protein loading. Lower panels, cell cycle distribution of control and SN38 treated (3μM, for 1h) CaSki and SiHa cells, analyzed 24, 48 and 72 h after treatment.

Mentions: Since both transcriptional and posttranslational mechanisms have been involved in the regulation of PLK1 expression [12, 29, 30], we investigated whether these regulatory processes could account for differences in PLK1 modulation in SCC cell lines. CPT-mediated transcriptional downmodulation of mitotic regulators, including PLK1, has been previously reported [31]. Quantitative RT-PCR results showed that PLK1 mRNA levels were decreased after 24h of SN38 treatment in both CaSki and SiHa cells (Fig. 2A). Immunoprecipitation of PLK1 from CaSki cells 6h after 1h of drug exposure evidenced a dose-dependent increase in the amount of ubiquitinated PLK1 (Fig. 2B). This finding was consistent with a functional G2/M DNA damage checkpoint promoting ubiquitination via cullin-based E3 ubiquitin ligases and subsequent proteasome-dependent degradation of crucial mitotic regulators such as the dual specificity phosphatase Cdc25A and PLK1 [11, 12, 30, 32]. Indeed, in CaSki cells, the downmodulation of PLK1 and Cdc25A was associated with increased levels of the mitosis markers phospho-Ser10 H3 and cyclin B1 (Fig. 2C). Analysis of cell cycle distribution evidenced in these cells a transient drug-induced accumulation of cells in G2/M phase preceding apoptosis induction (Fig. 2C and Suppl.Fig. 1B). In contrast, the lack of remarkable changes in the levels of PLK1 ubiquitination (Fig. 2B) and the absence of Cdc25A downmodulation observed in SiHa cells exposed to equimolar concentrations of SN38 (Fig. 2C), suggested a defective DNA damage checkpoint/signaling. Accordingly, the analysis of cell cycle distribution and of nuclear morphology evidenced a persistent drug-induced accumulation of SiHa cells with a 4N DNA content despite a low expression of cyclin B1 and phospho-Ser10 histone H3 (Fig. 2C and Suppl Fig. 1B). Furthermore, the hyperphosphorylation of RPA-2, associated with increased levels cyclin D1, indicated that SiHa cells had skipped the G2/M cell cycle phase, directly slipping into a G1-like phase despite the presence of persistent DNA damage. Similarly, in sarcoma cells, Cdc25A was downmodulated in the CPT-sensitive TC71 cells and not in RD cells in response to SN38 suggesting an impairment of DNA damage checkpoint in the resistant rhabdomyosarcoma cell line (Suppl. Fig. 1C). Moreover, Cdc25A appeared coherently modulated with PLK1 also in the in vivo setting in SCC from mice treated with CPT11 (Suppl. Fig. 1D). Thus, the failure to downregulate PLK1 through ubiquitilation and proteasome-dependent proteolysis appears to be associated with a less efficient CPT-induced G2/M checkpoint.


PLK1 is a critical determinant of tumor cell sensitivity to CPT11 and its inhibition enhances the drug antitumor efficacy in squamous cell carcinoma models sensitive and resistant to camptothecins.

Zuco V, De Cesare M, Zaffaroni N, Lanzi C, Cassinelli G - Oncotarget (2015)

Effects of SN38 treatment on PLK1 transcription and ubiquitination and on cell cycle distribution in SCC cell linesCaSki and SiHa cells were treated for 1h with solvent (−) or equimolar concentrations of SN38. A) Twenty four hours later, cells were analyzed for PLK1 mRNA expression by qRT-PCR. B) In parallel, six hours after the end of treatment (1h), cell lysates were subjected to PLK1 immunoprecipitation and analyzed by Western blotting with anti-ubiquitin antibody (in upper panel representative blots are shown). Histograms represent the densitometric quantification of bands corresponding to ubiquitinated relative to not ubiquitinated PLK1 in each cell lines. Data from two independent experiments are expressed in arbitrary units referred to untreated cells (lower panel). C) Cells were exposed to solvent (−) or to the indicated concentrations of SN38 for 1h. Cell lysates were analyzed by Western blotting to evaluate the levels of RPA-2, PLK1, Cdc25A and cell cycle-specific markers. The hyperphosphorylated form of RPA-2 in SiHa cells was evidenced by the electrophoretic mobility shift. Vinculin is shown as a control of protein loading. Lower panels, cell cycle distribution of control and SN38 treated (3μM, for 1h) CaSki and SiHa cells, analyzed 24, 48 and 72 h after treatment.
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Related In: Results  -  Collection

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Figure 2: Effects of SN38 treatment on PLK1 transcription and ubiquitination and on cell cycle distribution in SCC cell linesCaSki and SiHa cells were treated for 1h with solvent (−) or equimolar concentrations of SN38. A) Twenty four hours later, cells were analyzed for PLK1 mRNA expression by qRT-PCR. B) In parallel, six hours after the end of treatment (1h), cell lysates were subjected to PLK1 immunoprecipitation and analyzed by Western blotting with anti-ubiquitin antibody (in upper panel representative blots are shown). Histograms represent the densitometric quantification of bands corresponding to ubiquitinated relative to not ubiquitinated PLK1 in each cell lines. Data from two independent experiments are expressed in arbitrary units referred to untreated cells (lower panel). C) Cells were exposed to solvent (−) or to the indicated concentrations of SN38 for 1h. Cell lysates were analyzed by Western blotting to evaluate the levels of RPA-2, PLK1, Cdc25A and cell cycle-specific markers. The hyperphosphorylated form of RPA-2 in SiHa cells was evidenced by the electrophoretic mobility shift. Vinculin is shown as a control of protein loading. Lower panels, cell cycle distribution of control and SN38 treated (3μM, for 1h) CaSki and SiHa cells, analyzed 24, 48 and 72 h after treatment.
Mentions: Since both transcriptional and posttranslational mechanisms have been involved in the regulation of PLK1 expression [12, 29, 30], we investigated whether these regulatory processes could account for differences in PLK1 modulation in SCC cell lines. CPT-mediated transcriptional downmodulation of mitotic regulators, including PLK1, has been previously reported [31]. Quantitative RT-PCR results showed that PLK1 mRNA levels were decreased after 24h of SN38 treatment in both CaSki and SiHa cells (Fig. 2A). Immunoprecipitation of PLK1 from CaSki cells 6h after 1h of drug exposure evidenced a dose-dependent increase in the amount of ubiquitinated PLK1 (Fig. 2B). This finding was consistent with a functional G2/M DNA damage checkpoint promoting ubiquitination via cullin-based E3 ubiquitin ligases and subsequent proteasome-dependent degradation of crucial mitotic regulators such as the dual specificity phosphatase Cdc25A and PLK1 [11, 12, 30, 32]. Indeed, in CaSki cells, the downmodulation of PLK1 and Cdc25A was associated with increased levels of the mitosis markers phospho-Ser10 H3 and cyclin B1 (Fig. 2C). Analysis of cell cycle distribution evidenced in these cells a transient drug-induced accumulation of cells in G2/M phase preceding apoptosis induction (Fig. 2C and Suppl.Fig. 1B). In contrast, the lack of remarkable changes in the levels of PLK1 ubiquitination (Fig. 2B) and the absence of Cdc25A downmodulation observed in SiHa cells exposed to equimolar concentrations of SN38 (Fig. 2C), suggested a defective DNA damage checkpoint/signaling. Accordingly, the analysis of cell cycle distribution and of nuclear morphology evidenced a persistent drug-induced accumulation of SiHa cells with a 4N DNA content despite a low expression of cyclin B1 and phospho-Ser10 histone H3 (Fig. 2C and Suppl Fig. 1B). Furthermore, the hyperphosphorylation of RPA-2, associated with increased levels cyclin D1, indicated that SiHa cells had skipped the G2/M cell cycle phase, directly slipping into a G1-like phase despite the presence of persistent DNA damage. Similarly, in sarcoma cells, Cdc25A was downmodulated in the CPT-sensitive TC71 cells and not in RD cells in response to SN38 suggesting an impairment of DNA damage checkpoint in the resistant rhabdomyosarcoma cell line (Suppl. Fig. 1C). Moreover, Cdc25A appeared coherently modulated with PLK1 also in the in vivo setting in SCC from mice treated with CPT11 (Suppl. Fig. 1D). Thus, the failure to downregulate PLK1 through ubiquitilation and proteasome-dependent proteolysis appears to be associated with a less efficient CPT-induced G2/M checkpoint.

Bottom Line: Downregulation of the mitotic kinase PLK1 was found associated with apoptosis induced by SN38 (CPT11 active metabolite).The ability to activate an efficient G2/M cell cycle checkpoint allowing PLK1 ubiquitination and degradation was found associated with SN38-induced apoptosis in SCC cells.A well-tolerated CPT11/BI2536 cotreatment resulted in improved antitumor effect against SCC xenografts in mice compared to single agent treatments.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

ABSTRACT
Intrinsic and acquired tumor drug resistance limits the therapeutic efficacy of camptothecins (CPTs). Downregulation of the mitotic kinase PLK1 was found associated with apoptosis induced by SN38 (CPT11 active metabolite). We investigated the role of PLK1 in the cell response to CPTs in squamous cell carcinoma (SCC) and pediatric sarcoma cell lines and explored the therapeutic potential of the combination of CPT11 and the PLK1 inhibitor BI2536 in CPT-sensitive and -resistant tumor models. Gain- and loss-of-function experiments established a direct role for PLK1 in counteracting SN38 antiproliferative and pro-apoptotic effects. The ability to activate an efficient G2/M cell cycle checkpoint allowing PLK1 ubiquitination and degradation was found associated with SN38-induced apoptosis in SCC cells. However, the synergistic interaction between SN38 and BI2536 enhanced apoptosis in cell lines both sensitive and resistant to SN38-induced apoptotic cell death. A well-tolerated CPT11/BI2536 cotreatment resulted in improved antitumor effect against SCC xenografts in mice compared to single agent treatments. The increased apoptosis induction was reflected in a high rate of complete responses and cures in mice harboring SCC, including tumors with intrinsic or acquired resistance to CPTs. PLK1 inhibition represents a promising strategy to improve the antitumor efficacy of CPT11-based regimens.

No MeSH data available.


Related in: MedlinePlus