THOMAS LIEHR FIRST MOLECULAR CYTOGENETIC CHARACTERIZATION OF THE MMT 060562 MURINE BREAST CANCER CELL LINE Текст научной статьи по специальности «Биотехнологии в медицине»

Научные результаты биомедицинских исследований. 2021;7(1):4-14 Research Results in Biomedicine. 2021:7(1):4-14

ГЕНЕТИКА GENETICS

DOI: 10.18413/2658-6533-2020-7-1-0-1

First molecular cytogenetic characterization of the MMT 060562 murine breast cancer

cell line

Shaymaa Azawi , Lisa-Marie Barf , Thomas Liehr

Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Am Klinikum 1, D-07747 Jena, Germany Corresponding author: Thomas Liehr (Thomas.Liehr@med.uni-jena.de)

Abstract

Background: Murine cell lines are working horses applied as model systems in multiple research studies in many laboratories. Nonetheless, most of them are not well characterized at the genetic level. This diagnosis holds also true for the MMT 060562 murine breast cancer cell line, also referred to as MMT060562 or MMT-060562. The aim of the study: To provide detailed cytogenetic characterization of the MMT 060562 cancer cell line. Materials and methods: The cell line was studied by molecular cytogenetics, namely by fluorescence in situ hybridization applying all murine while chromosome paints in one probe set and all chromosome-specific murine multicolor banding probe sets. Results: For this we present here the first detailed karyotype of the established in 1962 cell line, a comprehensive map of chromosomal imbalances and an in-silico translation of the results to the human genome. Surprisingly, MMT 060562 has only few chromosomal aberrations, even a cell clone without any gross chromosomal abnormalities in ~40% of the cells, and with most aberrant of four cell clones showing a dicentric dic(2;17)(A1A1), a derivative del(3)(A3C), a trisomy 6, and a derivative

derQ4)t(13;14X14pter^14D1::14B^14D1::13A3^13qter). Conclusion: It could be shown, that MMT 060562 is most similar to human breast cancer of basal-like tumor type. Thus, this cell line can serve as a model for a very early breast cancer stage and thus closes a gap in the yet available cell lines.

Keywords: breast cancer (BC); MMT 060562 murine cell line; murine multicolor banding (mcb)

For citation: Azawi S, Barf LM, Liehr T. First molecular cytogenetic characterization of the MMT 060562 murine breast cancer cell line. Research Results in Biomedicine. 2021;7(1):4-14. DOI: 10.18413/2658-6533-2020-7-1-0-1

Introduction. In female breast cancer (BC), being a major leading cause of human cancer death, survival chance varies still tremendously, depending on many factors like awareness, preventive programs, diagnostic regimens, and possibilities to treat this disease [1]. The incidence of this malignancy is furthermore influenced by such factors as average life expectancy in the country the person is derived from, life style, individual estrogen-levels, number of children of the woman, and family history of cancer, including adverse gene mutations [1, 2]. As BC is heterogenic, it is divided into subtypes according to morphology, molecular profiles, and specific biomarker expression [3]. Accordingly, various subtypes may have different prognoses, clinical outcomes, and maybe in need of specific treatment regimens [3, 4]. Biomarkers studied to perform BC subclassifications may include oncogene and tumor suppressor gene expression levels, as well as that of other gene-products, like estrogen-, progesterone-, human epidermal growth factor-2- (HER-2/ ERBB2) and epidermal growth factor-receptor, or cy-tokeratin 5 or nuclear protein Ki67 [5, 6]. Thus, BC may be classified in (a) luminal Alike, (b) luminal B-like (HER2-positive and HER2-negative), (c) HER2-overexpressing, and (d) triple-negative subtypes [3, 6]. Furthermore, liquid biopsy plays an increasingly important role in BC follow-up [7].

Various treatment regimens for BC subtypes are available [6, 8], however, severe side effects cannot be excluded in many of these approaches. Also, aggressive courses of disease and limitations in BC treatment are not unusual

[9]. Therefore, research on BC-biology, as well as for new treatment strategies is imperative

[10], which are undertaken in cell cultures based on murine BC cell lines [5].

The MMT 060562 murine BC-cell line, also referred to as MMT060562 or MMT-060562, has been applied in about 2 dozen studies (see [11]), and, as most of other such cell lines [12], still been not characterized in detail genetically. According to ATCC, this cell line was established in 1962 [13] as being derived from a hybrid female C57BL/6xAf mouse as a spontaneous malignant neoplasm

of the mouse mammary gland [14]. To the best of our knowledge the karyotype of this cell line was never published before, however, according to ATCC, MMT 060562 has a modal number of 40 chromosomes with a range of 36 to 81, and a stem line number being diploid [13], while ECAAC gives a description that the cell line is diploid with 2n = 40 [14]. To close the gap in the literature, here we present the first molecular cytogenetic characterization of the MMT 060562 cell line.

Material and Methods

Cell line. The MMT 060562 cell line was purchased from the American Type Culture Collection (ATCCR CCL-51™; Wesel Germany) and grown adherently according to the company's instructions: the cells were cy-togenetically worked up as previously described [12].

Molecular cytogenetics. FISH was done as previously reported [15] applying whole chromosome paints ("SkyPaintTM DNA Kit M-10 for Mouse Chromosomes", Applied Spectral Imaging, Edingen-Neckarhausen, Germany) for multicolor-FISH (mFISH), and murine chromosome-specific multicolor banding (mcb) probe mixes for FISH-banding [16]. At least 30 metaphases were analyzed for each probe set (Zeiss Axioplan microscopy, equipped with ISIS software (MetaSystems, Altlussheim, Germany).

Data analyses. Imbalances and breakpoints of MMT 060562 were determined according to mcb data and aligned to human homologous regions using the Ensembl and the UCSC Genome Browser, as previously described [12]. The obtained data were compared to genetic changes known from human BCs as previously done [5].

Ethics Statement. According to the Ethical Committee (medical faculty) and the Animal Experimentation Commission of the Friedrich Schiller University, there are no ethical agreements necessary for studies involving murine tumor cell lines like MMT 060562.

Results. MMT 060562 is a cell line with nearly stable diploid karyotype, only few single cell aberrations were present, which are not reported here. The aberrations detected

could be divided in two clones, with clone 2 having three sub-clones.

— Clone 1 can be considered as the ancestor and has a completely normal murine karyotype representing 42% of the studied metaphases; karyotype: 40,XY.

— Clone 2, falling in three subclones, has as typical aberration in common an additional chromosome 6:

• clone 2a showed a trisomy of chromosome 6 and an inversion in a chromosome 2 (11.3% of the cells); karyotype: 41,XY,inv(2)(C3E5),+6;

• clone 2b1 (28.5% of the cells) acquired a complicated aberration involving chromosomes 13 and 14;

41,XY,+6,der( 14)t( 13;14)(14pter— 14D1::14 B—14D1::13A3 —13qter);

• clone 2b2 (18.2% of the cells) acquired an additional aberration involving chromosomes 2 and 17 and showed the karyo-type

40,XY,dic(2;17)(A1A1),del(3)(A3C),+6,der(1 4)t(13;14)(14pter- 14D1::14B—14D1::13A3 13qter) (Fig. 1).

FISH-data translated to CGH-data is summarized in Table 1 and Fig. 2A. An in-silico translation of those results to the human genome identified the corresponding homologous region in the human genome (Table 1, Fig. 2B).

* I

mm

* -

S m

p ■

Fig. 1. Murine multicolor banding (mcb) was applied on chromosomes of the MMT 060562 cell line - here the result for clone 2b2 is shown. This figure depicts the summary of 20 chromosome-specific FISH-experiments as typical pseudocolor banding. Derivative chromosomes consisting of different chromosomes are highlighted by frames and shown twice in this summarizing karyogram.

Beginning of Table 1

Homologous regions of the MMT 060562 mouse cell line CNVs, translated to human

Mouse

Human

Type of CNV

2A1-A3

2A3

2A3-B

2B-D

2D-E3

2E3-F1

2F1

2F1

2F1-H4

10p12.1

2q22.2-q13

9q33.2-q34.3

2q22.1-q32.1

11p14.2-q12.1

15q13.3-q21.2

2p11.2-q11.2

2q13

20p13-q13.33

gain

6A1

6A1

6A1-B2

6B2

6B2-B3

6B3-C1

6C1

6C1

6C1-D1

6D1

6D1

6D1-E

6E1-E3

6E3

6E3-F1

6F1

6F1-F3

6F3

6F3

6F3-G3

7p22.1-p21.3

7q21.2-q21.3

7q31.1-q36.1

7q36.1

7p15.3-p14.3

4q22.1-q22.3

4q27

1p31.3

2p13.3-p11.2

3p25.2-p25.1

3q21.2-q21.3

3p14.1-p12.3

3p26.3-p25.2

3q21.3-q22.1

10q11.21-q11.22

22q11.1-q11.21

12p13.33-p13.31

12p11.21

12p11.21

12p13.31-p11.21

gain

13A3-A5

13A5

13A5-B1

13B1

13B1

13B1-B2

13B3

13B3

13B3

13B3

13B3

13B3-C1

13C1-C3

13C3-D1

13D1-D2

13D2

13D2

6p25.3-p23 6p23-p22.3 9q22.1-q22.32 5q31.1-q31.2 5q35.2-q35.3 9q21.32-q21.33 8q22.1 9p11.2 9p13.1 9q12-q13 9q22.32-q22.33 5p15.33-p15.31 5q14.3-q15 5q13.2-q14.3 5q11.1-q13.2 5p12 1p11.2_

gain

End of Table 1

Homologous regions of the MMT 060562 mouse cell line CNVs, translated to human

Mouse Human Type of CNV

14B 3p25.1 gain

14B 10q11.2-q11.23

14C1 14q22.1-q23.1

14C1-C3 14q11.2-q12

14C3 13q12.11

14C3 13q12.12

14C3-D1 13q14.2

14D1 13q12.12

14D1 13q14.2-q14.3

14D1 13q12.13

17A1 6q25.2-q25.3 gain

17A1 6q25.3-q27

17A1 6q27

17A1-A2 6q27

17A2-A3 5q15-q21.1

17A3 5q35.1

17A3 6p21.32-p21.2

17A3 16p13.3

17A3-B1 21q22.3

17B1 6p22.1-p21.32

17B1 19p13.12

17B1 19p13.2

17B2-C 6p21.2-p12.3

17C 2q12.2-q12.3

17C 3p25.1-p24.3

17D 19p13.3

17D-E1 5q21.1-q22.1

17E1 18p11.32-p11.22

17E1-E5 2p23.2-p16.3

17E5 2p16.3-p16.2

17E5 18p11.32

3A3 3q26.2-q26.32 loss

3A3-B 3q26.32-q27.1

3B 9p11.2

3B 9p12

3B 9q13

3B-C 4q27-q31.1

14D1 8p23.1 loss

14D1-D2 8p21.3-p12

14D2-D3 13q14.11-q14.2

14D3-E4 13q14.3-q33.1

2A1 n.a. breakpoint

2C3 2q23 breakpoint

2E5 15q15 breakpoint

3A3 3q26.2 breakpoint

3C 4q31.1 breakpoint

14D1 13q14.1 breakpoint

17A1 n.a. breakpoint

Fig. 2. Copy number variations detected in the MMT 060562 cell line are summarized here with respect to a diploid-basic karyotype. Gains are shown as green bars and losses are red, and breaks are registered as arrows. (A) Imbalances found in the cell line depicted along a murine chromosome set; (B) Results translated and projected along the human chromosome set.

Comparison with literature. The corresponding homologous regions of the MMT 060562 cell line compared with the common imbalances in related to human BC [5, 17, 18] revealed copy number variations in 9 of 21 regions (43%) known to harbor oncogenes and tumor suppressor genes (Table 2). The here reported breakpoints of MMT 060562

compared to chromosomal breaks of human BC presented a congruency of 6%, only (Table 3). The genetic alterations in the cell line correlated with the molecular subtype for human BC as shown in Table 4. The results revealed the best correspondence between MMT 060562 and human BC subtype basal-like tumors (18%).

Table 2

Oncogenes and tumor suppresser genes of importance in BC according to the literature [17, 18] and their involvement in gains or loss of copy numbers

in the studied cell line

Oncogenes and tumor suppressor genes Gene loci in human MMT 060562

NRAS 1p22 or p13 no CNV

MSH2 2p22 gain

RAF1 3p25 gain

RAR02 3p24 no CNV

MLH1 3p21 no CNV

APC 5q21 gain

MYB 6q22-q23 no CNV

IGFII-R 6q26 gain

MYC 8q24 no CNV

CDKN2A (p16INK4) 9p21 no CNV

PTEN 10q23 no CNV

HRAS 11p15.5 no CNV

CCND1 11q13 no CNV

INT2 11q13 no CNV

ATM 11q22 no CNV

CDKN1B (p27kip1) 12p13 gain

KRAS2 12p12.1 gain

BRCA2 13q12 gain

RB1 13q14.2 gain

CDH1 (E-cadherin) 16q22 no CNV

TP53 (p53) 17p13 no CNV

ERBB2 17q21 no CNV

BRCA1 17q21 no CNV

SERPINB5 (maspin) 18q21 no CNV

STK11 (LKB1) 19p13 gain

SUM of concordance in CNVs of potentially affected regions 9/21

Note: CNV - copy number variant

Table 3

Breakpoints in MMT 060562 compared to the observed acquired breaks in human BCs according to the literature [5]. Concordances with human breakpoints

are highlighted in bold

Breakpoint acc. to human genome Human BC MMT 060562

1p33 + -

1q25.3 + -

2p23 - +

2q31.3 + -

3p26.1 + (+)

3p12.3 + -

3q21.3 + -

4q22.3 + -

4q26 + -

4q31.23 + (+)

5p14.2 + -

5q13.2 + -

5q14.3 + -

6q12 + -

8q23.3 + -

8q24.22 + -

9p24.2 + -

9p21 + -

10p11.21 + -

11p15.5 + -

12q12.1 + -

12q24.31 + -

13q21.2 + -

13q14.1 - +

14q32 + -

15q15 - +

16q13.3 + -

17p12 + -

17q21 + -

19p13.1 + -

20q13.3 + -

22q12.2 + -

SUM of concordance 2/32

Table 4

Copy number changes associated with molecular subtypes of human BC, according to [19],

with the copy number variants (CNVs) in the MMT 060562 cell line. _Concordances with human CNVs (in italics) are highlighted in bold_

DNA changes in BC Subtypes Human BC MMT 060562

HER2+

17q11.1~12 gain no CNV

17q21.31~23.2 gain no CNV

SUM of concordance 0/2

Basal-like tumors

4p15.31 loss no CNV

5q12.3~13.2 loss gain

5q33.1 loss no CNV

6p12.3 gain gain

6p21.1~23 gain gain

8q24.21~24.22 gain no CNV

10p12.33~14 gain no CNV

10q23.33 loss no CNV

12q13.13~13.3 loss no CNV

15q15.1 loss gain

15q21.1 loss gain

SUM of concordance 2/11

Luminal A

1q21.3~44 gain no CNV

16p13.12~13.13 gain no CNV

16q11.2~13 loss no CNV

16q22.1-24.1 loss no CNV

SUM of concordance 0/4

Luminal B

1p31.3 loss gain

8p21.2~23.1 loss no CNV

17q23.2 gain no CNV

SUM of concordance 0/3

Note: no CNV - no copy number variant

Discussion. Heterogeneity of BC is one of the reasons that its biology is overall still poorly understood. Correspondingly, basic research and studies testing new potential therapeutics are necessary [9; 20]. MMT 060562 is a murine BC cell line yet not characterized cytogenomically in detail. Thus, its reluctant use in research studies, according to PUBMED [11] only ~20 papers are published using this cell line, here we did the first detailed molecular cytogenetic study to close this gap. The same approach as previously undertaken for several other murine cell lines

was done for MMT 060562 [5, 12, 15, 16, 21-23].

The MMT 060562 cell line presents a normal karyotype in ~40% of the studied cells, which is surprising for an almost 60-year-old cell line. Most likely it would be an ideal candidate to be studied by sequencing, to find submicroscopic mutations leading cells on the path towards BC-malignization. Also it is striking that since its establishment MMT 060562 cells did not tertaploidize, as reported mainly for human cell lines [24] and ~50% of murine tumour cell lines [5, 12, 15,

16, 21-23]. To the best of our knowledge, the MMT 060562 cell line is the less chromosomal aberrant malignant cell line ever reported. However, it is definitely a cell line, which induced tumors in nude mice [25].

Conclusion. Overall and in conclusion, the MMT 060562 cell line is a very interesting model system for early human BC, which should be studied in more detail and applied in corresponding studies for new therapeutica.

Financial support

Supported by grant # 2013.032.1 of the Wilhelm Sander-Stiftung.

Conflict of interests

The authors have no conflict of interest to declare.

References

1. Shah R, Rosso K, Nathanson SD. Pathogenesis, prevention, diagnosis and treatment of breast cancer. World Journal of Clinical Oncology. 2014;5(3):283-298. DOI: https://doi.org/10.5306/wjco.v5.i3.283

2. Sun YS, Zhao Z, Yang ZN, et al. Risk factors and preventions of breast cancer. International Journal of Biological Sciences. 2017; 13(11): 1387-1397. DOI: https://doi.org/10.7150/ijbs.21635

3. Januskeviciené I, Petrikaitè V. Heterogeneity of breast cancer: The importance of interaction between different tumor cell populations. Life Sciences. 2019;239(1):117009. DOI: https://doi.org/ 10.1016/j.lfs.2019.117009

4. Rouzier R, Perou CM, Symmans WF, et al. Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clinical Cancer Research. 2005;11(16):5678-5685. DOI: https://doi.org/10.1158/1078-0432.CCR-04-2421

5. Azawi S, Liehr T, Rincic M, et al. Molecular cytogenomic characterization of the murine breast cancer cell lines C-127I, EMT6/P and TA3 Hauschka. International Journal of Molecular Sciences. 2020;21(13):4716. DOI: https://doi.org/10.3390/ijms21134716

6. Kikuchi-Koike R, Nagasaka K, Tsuda H, et al. Array comparative genomic hybridization analysis discloses chromosome copy number alterations as indicators of patient outcome in lymph node-negative breast cancer. BMC Cancer. 2019;19(1):521. DOI: https://doi.org/10.1186/s12885-019-5737-7

7. Alimirzaie S, Bagherzadeh M, Akbari MR. Liquid biopsy in breast cancer: A comprehensive review. Clinical Genetics. 2019;95(6):643-660. DOI: https://doi.org/10.1111/cge.13514

8. Yersal O, Barutca S. Biological subtypes of breast cancer: Prognostic and therapeutic implications. World Journal of Clinical Oncology. 2014;5(3):412-424. DOI: http://dx.doi.org/10.5306/wjco.v5.i3.412.

9. Feng Y, Spezia M, Huang S, et al. Breast cancer development and progression: Risk factors, cancer stem cells, signaling pathways, genomics, and molecular pathogenesis. Genes and Diseases. 2018;5(2):77-106. DOI: https://doi.org/10.1016Zj.gendis.2018.05.001

10.Raihan J, Ahmad U, Yong YK, et al. Regression of solid breast tumours in mice by Newcastle disease virus is associated with production of apoptosis relatedcytokines. BMC Cancer. 2019;19(1):315. DOI: https://doi.org/10.1186/s12885-019-5516-5

11.PUBMED [Internet]. Seach for MMT 060562, MMT060562 or MMT-060562 [cited 2021 Jan 22]. Available from: https://pubmed.ncbi.nlm.nih.gov

12. Rhode H, Liehr T, Kosyakova N, et al. Molecular cytogenetic characterization of two murine colorectal cancer cell lines. OBM Genetics. 2018;2(3):1803037. DOI: https://doi.org/10.21926/obm.genet.1803037

13.ATCC [Internet]. [cited 2021 Jan 22]. Available from: https://www.lgcstandards-atcc.org/Products/All/CCL-51.aspx?geo_country=de#history

14.ECACC [Internet]. [cited 2021 Jan 22]. Available from: https://www.phe-culturecollec-

tions.org.uk/products/celllines/generalcell/detail.js p?refId=90111911&collection=ecacc_gc

15.Kubicova E, Trifonov V, Borovecki F, et al. First molecular cytogenetic characterization of murine malignant mesothelioma cell line AE17 and in silico translation to the human genome. Current Bioinformatics. 2017;12(1): 11-18. DOI: https://doi.org/10.2174/157489361166616060616 4459

16. Leibiger C, Kosyakova N, Mkrtchyan H, et al. First molecular cytogenetic high resolution characterization of the NIH 3T3 cell line by mu-rine multicolor banding. Journal of Histochemis-try and Cytochemistry. 2013;61(4):306-312. DOI: https://doi.org/10.1369/0022155413476868

17. Hall JM, Zuppan PJ, Anderson LA, et al. Oncogenes and human breast cancer. American Journal of Human Genetics. 1989;44(4):577-584.

18.Lebok P, Roming M, Kluth M, et al. p16 overexpression and 9p21 deletion are linked to unfavorable tumor phenotype in breast cancer. Oncotarget. 2016;7(49):81322-81331. DOI: https://doi.org/10.18632/oncotarget.13227

19.Horlings HM, Lai C, Nuyten DS, et al. Integration of DNA copy number alterations and prognostic gene expression signatures in breast cancer patients. Clinical Cancer Research. 2010;16(2):651-663. DOI: https://doi.org/10.1158/1078-0432.CCR-09-0709

20.Ye Y, Qiu TH, Kavanaugh C, et al. Molecular mechanisms of breast cancer progression: Lessons from mouse mammary cancer models and gene expression profiling. Breast Disease. 2004;19(1):69-82. DOI: https://doi.org/10.3233/bd-2004-19109

21.Guja K, Liehr T, Rincic M, et al. Molecular cytogenetic characterization identified the murine B-cell lymphoma cell line A-20 as a model for sporadic Burkitt's lymphoma. Journal of Histochemistry and Cytochemistry. 2017;65(11):669-677. DOI: https://doi.org/10.1369/0022155417731319

22. Steinacker R, Liehr T, Kosyakova N, et al. Molecular cytogenetic characterization of two murine cancer cell lines derived from salivary gland. Biological Communications. 2018;63(4):243-255. DOI: https://doi.org/10.21638/spbu03.2018.403

23.Wahlbul E, Liehr T, Rincic M, et al. Cy-togenomic characterization of three murine malignant mesothelioma tumor cell lines. Molecular Cytogenetics. 2020;13(1):43. DOI: https://doi.org/10.1186/s13039-020-00511-4

24.Tan Z, Chu DZV, Chan YJA, et al. Mammalian cells undergo endoreduplication in response to lactic acidosis. Scientific Reports. 2018;8(1):2890. DOI: https://doi.org/10.1038/s41598-018-20186-7

25. Ono K, Akatsu T, Kugai N, et al. The effect of deletion of cyclooxygenase-2, prostaglandin receptor EP2, or EP4 in bone marrow cells on osteoclasts induced by mouse mammary cancer cell lines. Bone. 2003;33(5):798-804. DOI: https://doi.org/10.1016/s8756-3282(03)00264-3

Received 9 January 2021 Revised 12 February 2021 Accepted 14 February 2021

Information about the authors Shaymaa Azawi, Researcher at the Molecular Cytogenetics Laboratory, Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Jena, Germany, E-mail: shayma.alazawi@yahoo.com, OR-CID: 0000-0001-8681-1768. Lisa-Marie Barf, Researcher at the Molecular Cytogenetics Laboratory, Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Jena, Germany, E-mail: Lisa-Marie.Barf@uni-jena.de, OR-CID: 0000-0001-7535-2690. Thomas Liehr, PhD, PD, Head of Molecular Cytogenetics Laboratory, Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Jena, Germany, E-mail: Thomas.Liehr@med.uni-jena.de, ORCID: 0000-0003-1672-3054.