- Email: [email protected]
threonine kinase-encoding gene, in colorectal cancers
G Model
ARTICLE IN PRESS
PRP-51829; No. of Pages 2
Pathology – Research and Practice xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Correspondence Table 2 Summary of PLK2 expression and frameshift mutation in colorectal cancers.
Frameshift mutation and loss of expression of PLK2, a serine/threonine kinase-encoding gene, in colorectal cancers To the Editor Polo-like kinases (PLKs) are a family of serine-threonine kinases that regulate many a cellular processes including cell cycle and cell death [1]. PLK2 regulates centriole duplication along with PLK4 and is involved in the G2/M checkpoint in response to DNA damage as a direct target for transcriptional regulation by p53 [1,2]. Transcriptional silencing of PLK2 is common in many cancers [2]. Also, expression of PLK2 induces apoptosis and knock-down of PLK2 induces cell proliferation by inhibiting apoptosis [1,2]. Together, these data may suggest that PLK2 may act as a tumor suppressor gene (TSG). PLK2 mutation has been observed in many cancers at low prevalence (0.6% of gastric cancer and 0% of colorectal cancer (CRC)) (http://www.intogen.org/) [3]. However, PLK2 mutation has not been explored in CRCs focused on microsatellite instability (MSI) phenotype that comprises approximately 10–30% of CRCs [4]. In the human genome (http://genome.cse.ucsc.edu/), we observed a PLK2 mononucleotide repeat (T8) in a coding exon that might be altered by deletion or duplication mutation in high MSI (MSI-H) cancers and analyzed the repeat by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay. We used methacarn-fixed tissues of 101 CRCs with high MSI (MSI-H) and 45 CRCs with microsatellite stable (MSS) phenotypes. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection [5]. Radioisotope ([32 P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP as described previously [5]. We observed frameshift mutations in 11 of the 146 CRCs, which consisted of a type of deletion mutation in the T8 that would lead to termination of PLK2 protein translation (Table 1). The PLK2 mutation was found in MSI-H CRCs (11/101, 10.9%), but there was not any in MSS CRCs (0/45) (Fisher’s exact test, p = 0.015). Also, to see whether PLK2 protein expression was altered as well, we analyzed the expression in 101 CRC2 with MSI-H and 45 CRCs with MSS by immunohistochemistry as described previously [6]. Positive PLK2 immunostaining was observed in 93
p-value
All cases
Positive PLK2 expression (%)
MSI-H Total with PLK2 mutation without PLK2 mutation
101 11 90
60 (59.4) 1 (9.1) 59 (65.6)
<0.001
MSS Colorectal cancer Total with PLK2 mutation without PLK2 mutation
45 0 45
33 (73.3) 0 (0) 33 (73.3)
1.000
CRC: colorectal cancer, MSI-H: high microsatellite instability, MSS: stable microsatellite instability.
(63.7%) of the 146 CRCs (Table 2). Positive PLK2 immunostaining in MSI-H CRCs (60/101) was significantly lower than that in MSS CRCs (33/45) (Fisher’s exact test, p = 0.041). Ten of 11 CRCs with the PLK2 frameshift mutation exhibited negative PLK2 immunostaining. MSI-H cancers with the frameshift mutations showed lower PLK2 immunopositivity than those devoid of the mutations (Table 2). The immunopositivity in the CRCs carrying the frameshift mutations (1/11) was lower than that without the frameshift mutations (92/135) (p < 0.001). In this study, we attempted to find frameshift mutations in PLK2 gene, which may inactivate the TSG functions of PLK2. We found frameshift mutations of PLK2 in approximately 11% of CRCs with MSI-H. In addition, we discovered a significant difference of the mutation prevalence between the CRCs with MSI-H and MSS, indicating that the mutations were MSI-H-specific. Next, we analyzed the expression of PLK2 protein in CRC tissues and found a significantly lower expression of PLK2 in CRCs with the PLK2 frameshift mutation than those without the mutation. Together, our data may indicate that PLK2 gene is altered in CRCs by both somatic frameshift mutation in the mononucleotide repeat and expression loss of PLK2, and suggest that the PLK2 mutation might underlie the expression loss of PLK2. Several PLK inhibitors have been developed as anticancer agents that mainly target PLK1, based on the roles of PLK1 in cell cycle progression [7]. However, the various ATP-competitive PLK inhibitors in clinical development inhibit the activities of PLK2 and PLK3 as well, which are considered as TSGs. PLK2 not only plays a role in the control of cell cycle, but also acts as a mediator of apoptosis and cellular stress [1,2]. In the present study, we discovered that PLK2 might be inactivated in MSI-H CRCs, suggesting that PLK2
Table 1 Summary of PLK2 mutations in colorectal cancers. Gene
Location
Wild type
Mutation
MSI status of the mutation cases (n)
Incidence in MSI-H cancers (%)
Nucleotide change (predicted amino acid change)
PLK2
Exon 7
T8
T7
MSI-H (11)
Colorectal: 11/101 (10.9)
c.1004delT (p.Leu335CysfsX68)
MSI-H: high microsatellite instability. http://dx.doi.org/10.1016/j.prp.2017.06.011 0344-0338/© 2017 Elsevier GmbH. All rights reserved.
Please cite this article in press as: J.H. Lee, et al., Frameshift mutation and loss of expression of PLK2, a serine/threonine kinase-encoding gene, in colorectal cancers, Pathol. – Res. Pract (2017), http://dx.doi.org/10.1016/j.prp.2017.06.011
G Model PRP-51829; No. of Pages 2
ARTICLE IN PRESS Correspondence / Pathology – Research and Practice xxx (2017) xxx–xxx
2
could be a TSG in these cancers. In this sense, administration of a PLK inhibitor to MSI-H CRCs could possibly result in adverse effects. Thus, it is imperative that when using a PLK inhibitor, the biological roles of other PLKs like PLK2 and the mutation status in cancer cells need to be considered to improve treatment strategies against MSI-H CRCs. MSI CRCs, however, are the carcinomas with one of the highest mutational load in humans [4]. Therefore, it should further be validated whether the conclusion for therapeutic effects has functional and clinical implication in MSI CRC. Acknowledgement This study was supported by grants from National Research Foundation of Korea (2012R1A5A2047939 and 2017R1A2B2002314). References [1] F.A. Barr, H.H. Silljé, E.A. Nigg, Polo-like kinases and the orchestration of cell division, Nat. Rev. Mol. Cell Biol. 5 (2004) 429–440. [2] H.C. Reinhardt, M.B. Yaffe, Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response, Nat. Rev. Mol. Cell Biol. 14 (2013) 563–580. [3] C. Kandoth, M.D. McLellan, F. Vandin, K. Ye, B. Niu, C. Lu, et al., Mutational landscape and significance across 12 major cancer types, Nature 502 (2013) 333–339. [4] K. Imai, H. Yamamoto, Carcinogenesis and microsatellite instability: the interrelationship between genetics and epigenetics, Carcinogenesis 29 (2008) 673–680.
[5] E.J. Choi, N.J. Yoo, M.S. Kim, C.H. An, S.H. Lee, Putative tumor suppressor genes EGR1 and BRSK1 are mutated in gastric and colorectal cancers, Oncology 91 (2016) 289–294. [6] J.H. Lee, Y.J. Choi, E.M. Je, H.S. Kim, N.J. Yoo, S.H. Lee, Frameshift mutation of WISP3 gene and its regional heterogeneity in gastric and colorectal cancers, Hum. Pathol. 50 (2016) 146–152. [7] K. Strebhardt, Multifaceted polo-like kinases: drug targets and antitargets for cancer therapy, Nat. Rev. Drug Discov. 9 (2010) 643–660.
Ju Hwa Lee 1 Min Sung Kim 1 Nam Jin Yoo Sug Hyung Lee ∗ Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea ∗ Corresponding author at: Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Republic of Korea. E-mail address: [email protected] (S.H. Lee) 1
These authors contributed equally to this work. 12 May 2017
Please cite this article in press as: J.H. Lee, et al., Frameshift mutation and loss of expression of PLK2, a serine/threonine kinase-encoding gene, in colorectal cancers, Pathol. – Res. Pract (2017), http://dx.doi.org/10.1016/j.prp.2017.06.011
ARTICLE IN PRESS
PRP-51829; No. of Pages 2
Pathology – Research and Practice xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Correspondence Table 2 Summary of PLK2 expression and frameshift mutation in colorectal cancers.
Frameshift mutation and loss of expression of PLK2, a serine/threonine kinase-encoding gene, in colorectal cancers To the Editor Polo-like kinases (PLKs) are a family of serine-threonine kinases that regulate many a cellular processes including cell cycle and cell death [1]. PLK2 regulates centriole duplication along with PLK4 and is involved in the G2/M checkpoint in response to DNA damage as a direct target for transcriptional regulation by p53 [1,2]. Transcriptional silencing of PLK2 is common in many cancers [2]. Also, expression of PLK2 induces apoptosis and knock-down of PLK2 induces cell proliferation by inhibiting apoptosis [1,2]. Together, these data may suggest that PLK2 may act as a tumor suppressor gene (TSG). PLK2 mutation has been observed in many cancers at low prevalence (0.6% of gastric cancer and 0% of colorectal cancer (CRC)) (http://www.intogen.org/) [3]. However, PLK2 mutation has not been explored in CRCs focused on microsatellite instability (MSI) phenotype that comprises approximately 10–30% of CRCs [4]. In the human genome (http://genome.cse.ucsc.edu/), we observed a PLK2 mononucleotide repeat (T8) in a coding exon that might be altered by deletion or duplication mutation in high MSI (MSI-H) cancers and analyzed the repeat by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay. We used methacarn-fixed tissues of 101 CRCs with high MSI (MSI-H) and 45 CRCs with microsatellite stable (MSS) phenotypes. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosin-stained slides by microdissection [5]. Radioisotope ([32 P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP as described previously [5]. We observed frameshift mutations in 11 of the 146 CRCs, which consisted of a type of deletion mutation in the T8 that would lead to termination of PLK2 protein translation (Table 1). The PLK2 mutation was found in MSI-H CRCs (11/101, 10.9%), but there was not any in MSS CRCs (0/45) (Fisher’s exact test, p = 0.015). Also, to see whether PLK2 protein expression was altered as well, we analyzed the expression in 101 CRC2 with MSI-H and 45 CRCs with MSS by immunohistochemistry as described previously [6]. Positive PLK2 immunostaining was observed in 93
p-value
All cases
Positive PLK2 expression (%)
MSI-H Total with PLK2 mutation without PLK2 mutation
101 11 90
60 (59.4) 1 (9.1) 59 (65.6)
<0.001
MSS Colorectal cancer Total with PLK2 mutation without PLK2 mutation
45 0 45
33 (73.3) 0 (0) 33 (73.3)
1.000
CRC: colorectal cancer, MSI-H: high microsatellite instability, MSS: stable microsatellite instability.
(63.7%) of the 146 CRCs (Table 2). Positive PLK2 immunostaining in MSI-H CRCs (60/101) was significantly lower than that in MSS CRCs (33/45) (Fisher’s exact test, p = 0.041). Ten of 11 CRCs with the PLK2 frameshift mutation exhibited negative PLK2 immunostaining. MSI-H cancers with the frameshift mutations showed lower PLK2 immunopositivity than those devoid of the mutations (Table 2). The immunopositivity in the CRCs carrying the frameshift mutations (1/11) was lower than that without the frameshift mutations (92/135) (p < 0.001). In this study, we attempted to find frameshift mutations in PLK2 gene, which may inactivate the TSG functions of PLK2. We found frameshift mutations of PLK2 in approximately 11% of CRCs with MSI-H. In addition, we discovered a significant difference of the mutation prevalence between the CRCs with MSI-H and MSS, indicating that the mutations were MSI-H-specific. Next, we analyzed the expression of PLK2 protein in CRC tissues and found a significantly lower expression of PLK2 in CRCs with the PLK2 frameshift mutation than those without the mutation. Together, our data may indicate that PLK2 gene is altered in CRCs by both somatic frameshift mutation in the mononucleotide repeat and expression loss of PLK2, and suggest that the PLK2 mutation might underlie the expression loss of PLK2. Several PLK inhibitors have been developed as anticancer agents that mainly target PLK1, based on the roles of PLK1 in cell cycle progression [7]. However, the various ATP-competitive PLK inhibitors in clinical development inhibit the activities of PLK2 and PLK3 as well, which are considered as TSGs. PLK2 not only plays a role in the control of cell cycle, but also acts as a mediator of apoptosis and cellular stress [1,2]. In the present study, we discovered that PLK2 might be inactivated in MSI-H CRCs, suggesting that PLK2
Table 1 Summary of PLK2 mutations in colorectal cancers. Gene
Location
Wild type
Mutation
MSI status of the mutation cases (n)
Incidence in MSI-H cancers (%)
Nucleotide change (predicted amino acid change)
PLK2
Exon 7
T8
T7
MSI-H (11)
Colorectal: 11/101 (10.9)
c.1004delT (p.Leu335CysfsX68)
MSI-H: high microsatellite instability. http://dx.doi.org/10.1016/j.prp.2017.06.011 0344-0338/© 2017 Elsevier GmbH. All rights reserved.
Please cite this article in press as: J.H. Lee, et al., Frameshift mutation and loss of expression of PLK2, a serine/threonine kinase-encoding gene, in colorectal cancers, Pathol. – Res. Pract (2017), http://dx.doi.org/10.1016/j.prp.2017.06.011
G Model PRP-51829; No. of Pages 2
ARTICLE IN PRESS Correspondence / Pathology – Research and Practice xxx (2017) xxx–xxx
2
could be a TSG in these cancers. In this sense, administration of a PLK inhibitor to MSI-H CRCs could possibly result in adverse effects. Thus, it is imperative that when using a PLK inhibitor, the biological roles of other PLKs like PLK2 and the mutation status in cancer cells need to be considered to improve treatment strategies against MSI-H CRCs. MSI CRCs, however, are the carcinomas with one of the highest mutational load in humans [4]. Therefore, it should further be validated whether the conclusion for therapeutic effects has functional and clinical implication in MSI CRC. Acknowledgement This study was supported by grants from National Research Foundation of Korea (2012R1A5A2047939 and 2017R1A2B2002314). References [1] F.A. Barr, H.H. Silljé, E.A. Nigg, Polo-like kinases and the orchestration of cell division, Nat. Rev. Mol. Cell Biol. 5 (2004) 429–440. [2] H.C. Reinhardt, M.B. Yaffe, Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response, Nat. Rev. Mol. Cell Biol. 14 (2013) 563–580. [3] C. Kandoth, M.D. McLellan, F. Vandin, K. Ye, B. Niu, C. Lu, et al., Mutational landscape and significance across 12 major cancer types, Nature 502 (2013) 333–339. [4] K. Imai, H. Yamamoto, Carcinogenesis and microsatellite instability: the interrelationship between genetics and epigenetics, Carcinogenesis 29 (2008) 673–680.
[5] E.J. Choi, N.J. Yoo, M.S. Kim, C.H. An, S.H. Lee, Putative tumor suppressor genes EGR1 and BRSK1 are mutated in gastric and colorectal cancers, Oncology 91 (2016) 289–294. [6] J.H. Lee, Y.J. Choi, E.M. Je, H.S. Kim, N.J. Yoo, S.H. Lee, Frameshift mutation of WISP3 gene and its regional heterogeneity in gastric and colorectal cancers, Hum. Pathol. 50 (2016) 146–152. [7] K. Strebhardt, Multifaceted polo-like kinases: drug targets and antitargets for cancer therapy, Nat. Rev. Drug Discov. 9 (2010) 643–660.
Ju Hwa Lee 1 Min Sung Kim 1 Nam Jin Yoo Sug Hyung Lee ∗ Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea ∗ Corresponding author at: Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Republic of Korea. E-mail address: [email protected] (S.H. Lee) 1
These authors contributed equally to this work. 12 May 2017
Please cite this article in press as: J.H. Lee, et al., Frameshift mutation and loss of expression of PLK2, a serine/threonine kinase-encoding gene, in colorectal cancers, Pathol. – Res. Pract (2017), http://dx.doi.org/10.1016/j.prp.2017.06.011