The Expression Pattern of Epidermal Differentiation Marker Keratin 10 in the Normal Human Breast and Breast Cancer Cells

Abstract

Cells of the human breast gland express an array of keratins, of which some are used for characterizing both normal and neoplastic breast tissue. However, the expression pattern of certain keratins has yet to be detailed. Here, the expression of a differentiation marker of epidermal epithelium, keratin 10 (K10), was investigated in the human breast gland. While in normal breast tissue generally less than 1% of luminal epithelial cells expressed K10, in women >30 years of age glandular structures with K10-positive (K10^pos) cells were found at higher frequency than in younger women. K10^pos cells belong to a mature luminal compartment as they were negative for cKIT, positive for Ks20.8, and mostly non-cycling. In breast cancer, around 16% of primary breast carcinomas tested were positive for K10 by immunohistochemistry. Interestingly, K10^pos tumor cells generally exhibit features of differentiation similar to their normal counterparts. Although this suggests that K10 is a marker of tumor differentiation, data based on gene expression analysis imply that high levels of K10 dictate a worse outcome for breast cancer patients. These findings can form the basis of future studies that should unravel which role K10 may play as a marker of breast cancer:

Keywords

estrogen receptor immunohistochemistry KRT10 Ks20.8 mammary gland

Introduction

The female human breast gland consists of an epithelium organized in a differentiation hierarchy that has yet to be fully understood. Unraveling the organization of this hierarchy is complicated by the fact that several events can lead to changes in the composition of the gland with increased age, including repeated cycles of menstruation, pregnancy and lactation, as well as postmenopausal regression. Acquiring a detailed insight into the various cellular subsets that populate the epithelial compartment in the normal gland may be key to understand the mechanisms that underlie the development of different breast cancer subtypes.

The distinct expression patterns of keratin intermediary filaments have widely been used to characterize the epithelia of various organs. In a number of epithelia, a subcellular change of keratin expression marks a transit between differentiation states. For example, in the epidermis there is a switch from expression of K5 and K14 in cells of the basal layer that provide the stem cell compartment to K1 and K10 in the differentiating suprabasal keratinocytes.¹ Thus, a distinct distribution of keratins within an epithelium may be involved in compartmentalizing various differentiation states.

The luminal epithelial cells that line the ducts and lobular ductules in the human breast gland express a selection of keratins that characterize simple epithelia, including K7,² K8,³ K18,³ and K19.⁴ However, the expression pattern is more complex as subsets of cells also express several keratins characteristic of stratified epithelia, including K5,⁵ K6a,⁶ K14,⁷ K15,⁶ and K17.⁷ Documentation of the presence of other stratified epithelial keratin markers in the breast gland is limited.⁸ For K10, its expression has not been described in the normal gland, and in breast cancer it has been reported to be present in rare cases.^9–11

This study reveals that in fact rare K10^pos cells are found in the normal human breast gland and that structures that contain these cells are more frequently present in women >30 years of age. Marker expression analysis demonstrated that K10^pos cells are part of a differentiated compartment in both normal breast and breast cancer tissue, and that a significant subset of tumors are in fact K10^pos.

Materials and Methods

Human Breast Tissue Samples

Normal human breast tissue was donated by consenting women undergoing reduction mammoplasty for cosmetic reasons performed at Capio CFR Hospitaler at Lyngby or Hellerup, Denmark. All personal information on donors was confidential except for the date and year of birth. In some cases, only the year of birth was available. Material from some of the biopsies has been used in previous studies.^12–15 Samples of breast cancer were obtained completely anonymously from patients at the State University Hospital, Copenhagen. The storage and use of human material have been reviewed and approved by the Regional Scientific Ethical Committees (H-2-2011-052 and H-3-2010-095) and the Danish Data Protection Agency (2011-41-6722) and have been handled according to established guidelines in subsequent experiments.

Immunostaining

Cryopreserved tissue specimens were cut at 6 μm on a cryostat (Microm HM560; Axlab, Vedbæk, Denmark) and were subsequently fixed either in ice cold methanol for 5 min and air-dried or in 3.7% formaldehyde (Merck; Darmstadt, Germany), followed by permeabilization with Triton X-100 detergent (Sigma-Aldrich, Denmark). For peroxidase staining, specimens were incubated with primary antibody for 1 hr, and Ultravision ONE Detection System was used according to the manufacturer’s instructions (Labvision; Thermo Fisher Scientific, Denmark). Nuclei were stained with hematoxylin. For immunofluorescence, the specimens were incubated for 2 hr with primary antibody followed by incubation with secondary isotype-specific Alexa Fluor antibodies for 30 min. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Cultured cells were fixed and stained as described above. For staining of smears, cells were first dissociated to single cells from breast epithelial organoids isolated as described previously.^12,16 Apart from classifying them by the presence of estrogen receptor α (ERα), breast carcinomas were also subdivided into molecular phenotypes by staining for progesterone receptor and HER2: Luminal A (ERα^pos and/or PR^pos, no HER2 overexpression), Luminal B (ERα^pos and/or PR^pos, HER2 overexpression), HER2-enriched (ERα^neg, PR^neg, HER2 overexpression), and Triple-Negative (ERα^neg, PR^neg, no HER2 overexpression). Antibodies and dilutions are listed in Table 1.

Table 1.

List of Antibodies Used for Immunostaining.

Antibody Target	Clone	Company/Cat. No.	Peroxidase	Fluorescence
cKIT	K45	NeoMarkers/MS-289	1:100	1:25
Estrogen receptor α	1D5	Dako/M7047	1:100	—
HER2	TAB250	Invitrogen/28-0003Z	1:200	—
Keratin 10	DE-K10	Invitrogen/MA5-13705	1:50	1:25
Keratin 10	RKSE60	Millipore/MAB3230	1:10	—
Keratin Ks20.8	Ks20.8	Dako/M7019	—	1:10
Keratin	CAM5.2	BD Biosciences/345779	—	1:25
Ki67	SP6	Thermo Scientific/RM-9106	—	1:25
Progesterone receptor	Pgr636	Dako/M3569	1:100	—
Alexa Fluor 488 goat anti-mouse IgG1	—	Invitrogen/A21151	—	1:500
Alexa Fluor 568 goat anti-mouse IgG2a	—	Invitrogen/A21134	—	1:500
Alexa Fluor 568 goat anti-rabbit IgG	—	Invitrogen/A11036	—	1:500

Microscopy and Image Acquisition

A lobular or ductal segment with the presence of one or more K10-expressing cells after performing immunohistochemistry (IHC) was assigned as K10^pos. For breast carcinomas, a tumor was considered positive for K10, cKIT, Ks20.8, ERα, or PR when >1% of the neoplastic cells expressed the given marker. Quantification of smear stainings and sections stained for Ki67 was performed on images that were acquired and digitally stored. Images were acquired on a Leica DM550B microscope equipped with a DFC550 digital camera or a Zeiss LSM 700 confocal microscope located at the Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen. In smears, 1000 CAM5.2-positive (CAM5.2^pos) luminal cells were quantified for each biopsy. For quantification of Ki67-positive cells in cultures, 200 cells were counted (either within the K10^pos population or on the overall population) in each of three different culture flasks. On tissue sections, 200 K10^pos cells or CAM5.2^pos cells were evaluated for Ki67 expression for each of three tumors.

Cell Culture

MCF10A is a spontaneously immortalized breast epithelial–derived non-tumorigenic cell line.¹⁷ MCF10A cells were cultured in Dulbecco’s Modified Eagle’s Medium/Nutrient mixture F-12 (DMEM/F12, 1:1; Life Technologies, Denmark) supplemented with 2 mmol/l glutamine, 20 ng/ml epidermal growth factor (PeproTech, Germany), 10 µg/ml insulin, 0.5 µg/ml hydrocortisone, 10 ng/ml cholera toxin (Sigma-Aldrich), and 5% fetal bovine serum (FBS). MCF7 breast cancer cells were cultured in DMEM 1965 supplemented with 2 mmol/l glutamine, 7 non-essential amino acids (NEA), 6 ng/ml insulin (Sigma-Aldrich), and 5% FBS. All cells were cultured at 37C in a humidified incubator with an atmosphere of 5% CO₂.

RNA Isolation and Real-time Quantitative PCR

For each biopsy, 10 tissue slices with a thickness of 6 to 8 µm were sectioned on a cryostat and dissolved in Trizol (Life technologies). Total RNA was isolated by a spin column method (Zymo Research, CA). The quality and integrity of the extracted RNA was evaluated by a spectrophotometer (Nanodrop 1000). RNAs were reverse transcribed using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Denmark). Quantified real-time PCR was performed using Taqman gene expression assays (Applied Biosystems) on Bio-Rad CFX manager 3.0 and thermocycler (Bio-Rad, Denmark). Expression of KRT10 (Hs00166289_m1) was normalized to GAPDH (Hs02758991_g1) and EPCAM (Hs00901885_m1).

Kaplan–Meier Plots

A web tool, KM-plotter (kmplot.com), was used to generate Kaplan–Meier plots based on data sets from more than 6000 breast cancer patients.¹⁸ Analysis of expression of KRT10 in the data sets was performed using the JetSet best probe set (Affymetrix ID: 213287_s_at). Expression data were trichotomized to compare the patients with the highest levels of KRT10 tumor expression (upper tertile) vs the lowest (lower tertile) for relapse-free survival (RFS) and overall survival (OS). Patients with ERα-positive and ERα-negative tumors were analyzed separately, as well as in total. Furthermore, more detailed subcluster analysis was performed by including parameters such as molecular subtype, tumor grade, lymph node status, and treatment modality. The number of patients analyzed can be found in Supplemental Table S4.

Results

Rare K10^pos Luminal Cells Are Most Frequently Present in Breast Gland Structures of Women >30 Years of Age

Here, we analyzed the expression of K10 in human normal breast glands by IHC on cryostat sections using a well-characterized antibody, clone DE-K10.^19,20 A minor subset of glandular structures demonstrated the presence of K10^pos cells. Typically, when present, K10^pos cells were located as single cells or in small foci as part of the luminal compartment (Fig. 1A). The staining pattern was validated by comparison with another often-used monoclonal antibody against K10, RKSE60,^21–23 in serial sections (Supplemental Fig. S1). The actual proportion of luminal cells expressing K10 was quantified on trypsinized cellular smears derived from breast gland tissue from five women (age range: 19–46 years). In all cases, less than 1% of luminal cells were positive (Supplemental Fig. S2).

To obtain a more comprehensive expression pattern of K10 in the normal breast gland, tissue sections from 45 women aged 13 to 74 years were immunostained. A total of 956 glandular structures were evaluated, of which 54 (5.6%) contained K10^pos cells (Supplemental Table S1). Overall, K10^pos structures showed a fairly even distribution between lobules (38 of 604 lobules, 6.3%) and ducts (16 of 352 ducts, 4.5%) (Table 2; Supplemental Table S1).

Table 2.

Expression of K10 in the Normal Breast Gland by Immunohistochemistry.

Fraction of Structures With K10^pos Cells
	n	%
Lobules	38/604	6.3
Ducts	16/352	4.5
Total	54/956	5.6

Abbreviation: K10^pos, K10-positive.

Dividing the biopsies into two age groups revealed that for women <30 years, 3 of 23 biopsies (13.0%) had K10^pos structures, whereas they were present in 10 of 23 biopsies (43.5%) from women >30 years (Fig. 1B, p<0.05 by Pearson’s Chi-square test).

Thus, rare K10^pos luminal cells are more frequently found in normal breast gland structures in women >30 years of age.

K10^pos Cells Belong to a Mature Luminal Compartment

We have previously used markers that define various differentiation states within the luminal lineage by IHC.^6,13,24 Whereas cKIT defines luminal progenitor cells,^14,24–26 Ks20.8 is a marker that encompasses a mature luminal compartment expressing ERα, GATA3, and CD166.¹³

In tissue sections double-stained for K10 and cKIT by immunofluorescence, the two markers generally segregated into different cells (Fig. 2A). Moreover, when staining for K10 and Ks20.8, the two markers co-stained, albeit with Ks20.8 more widely present in luminal cells (Fig. 2B). Furthermore, no K10^pos cells were found to express the cell cycle marker Ki67 (Fig. 2C). Due to the low frequency of K10^pos cells in the tissue sections, it was not possible to estimate the actual frequency of cells in cycle in comparison with luminal cells in general. Instead, a normal-derived breast epithelial cell line MCF10A containing a minor population of K10^pos cells was tested. Although around 88% of MCF10A cells in general were cycling by evaluating Ki67, only around 9% of K10^pos cells expressed Ki67 (Supplemental Fig. S3).

The lack of cKIT, the presence of Ks20.8, and the low number of cycling K10^pos cells suggest that these cells belong to a subset of mature luminal cells in the normal breast gland.

K10^pos Breast Carcinomas Mimic a Normal Luminal Differentiation Pattern

As carcinomas often reflect the epithelium of origin with regard to keratin expression,¹ we stained a sample of breast carcinomas for K10 (Fig. 3B). In total, 22 of 135 tumors (16.3%) were positive (Fig. 3B; Supplemental Table S2). When subdividing the stained carcinomas into ERα-positive and ERα-negative, respectively, 17 of 84 (20.2%) and 5 of 51 (9.8%) were K10^pos (Fig. 3B; Supplemental Table S2). Further classification of tumors into molecular phenotypes revealed that 13 of 74 (17.6%) Luminal A, 4 of 10 (40%) Luminal B, 1 of 16 (6.3%) HER2-enriched, and 4 of 35 (11.4%) Triple-Negative were K10^pos. Detection of K10 by IHC was further corroborated by its gene expression in breast tumors verified by real-time quantitative reverse transcriptase PCR (Fig. 4).

To analyze whether K10^pos cells in breast tumors retain a phenotype mimicking a mature luminal differentiation program found in normal tissue, we verified the expression of cKIT and Ks20.8 in all K10^pos tumors. Nineteen of 22 tumors were negative for luminal progenitor marker cKIT (Supplemental Table S3). None of the three cKIT-positive tumors coexpressed K10 and cKIT when tested by immunofluorescence (Fig. 5A).

Of the 22 K10^pos tumors, 21 (95%) also expressed the mature luminal marker Ks20.8 (Supplemental Table S3). Testing a sample of four Ks20.8-positive tumors revealed that K10^pos cells generally also expressed Ks20.8 (Fig. 5B).

Furthermore, the presence of cell cycle marker Ki67 was less frequent in K10^pos cells compared with the overall fraction of cycling neoplastic cells when performing quantification on three tumor biopsies (Supplemental Fig. S4A–C). Immunostaining of breast cancer cell line MCF7 revealed a similar pattern (Supplemental Fig. S4D).

Overall, K10^pos cells in breast carcinomas retain features that resemble mature luminal cells of the normal breast gland.

Expression of KRT10 Correlates to a Worse Outcome for Breast Cancer Patients

Using a web-based tool and publicly available data,²⁷ we compared breast cancer patients with the highest and lowest levels of expression of the K10 gene, KRT10. Surprisingly, the data revealed that when compared with low levels of KRT10 in breast tumors, high levels of KRT10 correlated to a worse RFS as well as OS for breast cancer patients (Fig. 6; Supplemental Table S4A and B). This correlation was evident for both ERα-positive and ERα-negative tumors. Further subsegmentation of the available data revealed similar correlation within the Luminal A subtype, for grade 3 tumors, and for lymph node–positive tumors that were ER-negative (Supplemental Fig. S5 and Table S4C).

Thus, transcriptome analysis suggests that K10 is a marker for poor prognosis in breast cancer.

Discussion

In epidermis, K10 is a marker of cells that exit cell cycle to enter terminal differentiation.^28,29 Thus, it is tempting to speculate that the presence of K10 in cells of the breast gland reflects a corresponding placement in a differentiation hierarchy. The features of K10^pos luminal cells of the breast do support a placing in a similar differentiated compartment, as they are negative for cKIT, positive for Ks20.8, and generally non-cycling. However, the low frequency of K10^pos cells can also lead to speculations that formation of these cells is an aberrant reminiscence of epidermal differentiation rather than a functional part of the normal differentiation program of the breast gland. In culture-based assays, myoepithelial cells derived from normal breast glands can undergo squamous cell differentiation, expressing K10.^14,30 Although rare, squamous cell metaplasia can also be found in the breast gland, often with prominent histological features.^31,32 However, when identified in the sample set of normal-derived biopsies that was analyzed in this study, K10^pos cells did not accumulate in characteristic metaplastic structures. Rather, K10^pos cells manifested themselves as infrequent single cells or distinct foci embedded as part of the normal luminal layer of glandular structures. Further investigations are required to determine whether the presence of K10 correlates to underlying metaplastic changes.

As age is the only disclosed information on the donors of the breast tissue in this study, other possible confounding events such as parity cannot fully be taken into account. In Denmark, the average age of primiparous women is 29 years,¹² so the presence of an increased frequency of K10^pos breast gland structures in women >30 years of age may be a reflection of age-related accumulation, of parity, or both. Although the majority of women with K10^pos breast gland structures were in the age range of 43 to 53 years, the present material does not allow us to conclude whether the frequency drops in postmenopausal women.

Although it has been suggested that K10 can be expressed in breast cancer,¹⁰ previously published studies indicate that it is a rare event at most.^9,33,34 A more recent reporting on K10 expression in breast tumors was only concerning rare types of carcinomas with clear squamous differentiation.¹¹ The fact that overall around 16% of breast carcinomas in our sample set were K10^pos clearly demonstrates that the expression of K10 is not confined to metaplastic tumors. The relatively frequent observation of K10^pos carcinomas by immunohistochemical stainings may relate to our use of cryosections rather than paraffin-embedded tissue, which could reduce antigen masking. Interestingly, K10^pos tumors were found in both ERα-positive and ERα-negative subgroups. However, as all but one of the 22 K10^pos carcinomas were also positive for luminal differentiation marker Ks20.8, it could be indicative of a certain stage of differentiation. Although based on a very small sample set of only 10 tumors, it does stand out that 40% of Luminal B carcinomas were K10^pos.

As the expression of K10 correlates to a more differentiated phenotype in normal tissue, it is somewhat surprising that apparently breast cancer with high KRT10 expression generally leads to a worse outcome than tumors with low levels of KRT10. We have previously reported on the presence of increased tumor-initiating capacity in a more differentiated subset of cells within a cancer stem cell hierarchy.^35,36 Also, the fact that most of K10^pos tumors demonstrate considerable intratumoral heterogeneity with respect to this marker may reflect the phenotypic plasticity of the tumor cells rather than the differentiation level of the tumor per se. It should be noted that in hepatocellular carcinoma it has been shown that K10 actually is a marker for poor prognosis.³⁷ Based on this, we suggest that further investigations on the role of K10 in breast cancer are initiated.

Supplemental Material

2019-00222R1_Production_Supplemental_Material_online_supp – Supplemental material for The Expression Pattern of Epidermal Differentiation Marker Keratin 10 in the Normal Human Breast and Breast Cancer Cells

Supplemental material, 2019-00222R1_Production_Supplemental_Material_online_supp for The Expression Pattern of Epidermal Differentiation Marker Keratin 10 in the Normal Human Breast and Breast Cancer Cells by Jiyoung Kim and René Villadsen in Journal of Histochemistry & Cytochemistry

Footnotes

Acknowledgements

We thank Lena Kristensen and Anita Sharma Friismose for expert technical assistance. Vera Timmermans, Pathology Department, Rigshospitalet, is acknowledged for providing tumor biopsies, and Benedikte Thuesen, Capio CFR Hospitaler, is acknowledged for providing normal breast biopsy material. The Core Facility for Integrated Microscopy (University of Copenhagen) is acknowledged for confocal microscope accessibility.

Competing Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Author Contributions

RV conceived the project, designed the study, performed the experiments, analyzed the data, and wrote the manuscript. JK designed the study, performed the experiments, analyzed the data, and wrote the manuscript.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Anita og Tage Therkelsens Fond, Læge Sophus Carl Emil Friis og hustru Olga Friis’ Legat, Oda og Hans Svenningens Fond, Tora og Viggo Groves Mindelegat, and Kong Christian den Tiendes Fond (to R.V.) and by Familien Erichsens Mindefond and Vera og Carl Johan Michaelsens Legat (to J.K.).

References

Moll

Divo

Langbein

The human keratins: biology and pathology. Histochem Cell Biol. 2008;129:705–33.

Ramaekers

Huysmans

Schaart

Moesker

Vooijs

Tissue distribution of keratin 7 as monitored by a monoclonal antibody. Exp Cell Res. 1987;170:235–49.

Lane

EB.

Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization. J Cell Biol. 1982;92:665–73.

Bartek

Taylor-Papadimitriou

Miller

NMR

. Patterns of expression of keratin 19 as detected with monoclonal antibodies in human breast tissues and tumours. Int J Cancer. 1985;36:299–306.

Boecker

Buerger

. Evidence of progenitor cells of glandular and myoepithelial cell lineages in the human adult female breast epithelium: a new progenitor (adult stem) cell concept. Cell Prolif. 2003;36(Suppl 1):73–84.

Villadsen

Fridriksdottir

Rønnov-Jessen

Gudjonsson

Rank

LaBarge

Bissell

Petersen

OW.

Evidence for a stem cell hierarchy in the adult human breast. J Cell Biol. 2007;177:87–101.

Wetzels

Kuijpers

Lane

Leigh

Troyanovsky

Holland

van Haelst

Ramaekers

FC.

Basal cell-specific and hyperproliferation-related keratins in human breast cancer. Am J Pathol. 1991;138:751–63.

Moll

Cytokeratins as markers of differentiation in the diagnosis of epithelial tumors. Subcell Biochem. 1998;31:205–62.

Heatley

Maxwell

Whiteside

Toner

Cytokeratin intermediate filament expression in benign and malignant breast disease. J Clin Pathol. 1995;48:26–32.

10.

Naz

Yang

Zhou

Xue

Breast cancer: search of hidden culprits. Breast J. 2010;16:215–7.

11.

Boecker

Stenman

Loening

Andersson

Berg

Lange

Bankfalvi

Samoilova

Tiemann

Buchwalow

Squamous/epidermoid differentiation in normal breast and salivary gland tissues and their corresponding tumors originate from p63/K5/14-positive progenitor cells. Virchows Arch. 2015;466:21–36.

12.

Ísberg

ÓG

Kim

Fridriksdottir

Morsing

Timmermans-Wielenga

Rønnov-Jessen

Petersen

Villadsen

. A CD146 FACS protocol enriches for luminal keratin 14/19 double positive human breast progenitors. Sci Rep. 2019;9:14843.

13.

Fridriksdottir

Klitgaard

Kim

Hopkinson

Petersen

Rønnov-Jessen

Propagation of oestrogen receptor-positive and oestrogen-responsive normal human breast cells in culture. Nat Commun. 2015;6:8786.

14.

Fridriksdottir

Villadsen

Morsing

Klitgaard

Kim

Petersen

Rønnov-Jessen

Proof of region-specific multipotent progenitors in human breast epithelia. Proc Natl Acad Sci U S A. 2017;114:E10102–11.

15.

Balk-Møller

Kim

Hopkinson

Timmermans-Wielenga

Petersen

Villadsen

A marker of endocrine receptor-positive cells, CEACAM6, is shared by two major classes of breast cancer: luminal and HER2-enriched. Am J Pathol. 2014;184:1198–208.

16.

Rønnov-Jessen

Petersen

OW.

Induction of alpha-smooth muscle actin by transforming growth factor-beta 1 in quiescent human breast gland fibroblasts. Implications for myofibroblast generation in breast neoplasia. Lab Invest. 1993;68:696–707.

17.

Soule

Maloney

Wolman

Peterson

Brenz

McGrath

Russo

Pauley

Jones

Brooks

SC.

Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10. Cancer Res. 1990;50:6075–86.

18.

Nagy

Lánczky

Menyhárt

Gyorffy

. Validation of miRNA prognostic power in hepatocellular carcinoma using expression data of independent datasets. Sci Rep. 2018;8:9227.

19.

Ivanyi

Ansink

Groeneveld

Hageman

Mooi

Heintz

APM

. New monoclonal antibodies recognizing epidermal differentiation-associated keratins in formalin-fixed, paraffin-embedded tissue. Keratin 10 expression in carcinoma of the vulva. J Pathol. 1989;159:7–12.

20.

Borowiec

Delcourt

Dewailly

Bidaux

. Optimal differentiation of in vitro keratinocytes requires multifactorial external control. PLoS ONE. 2013;8:e77507.

21.

Ramaekers

Puts

Moesker

Kant

Huysmans

Haag

Jap

Herman

Vooijs

GP.

Antibodies to intermediate filament proteins in the immunohistochemical identification of human tumours: an overview. Histochem J. 1983;15:691–713.

22.

Berge

Kristensen

Rattan

SIS

. Kinetin-induced differentiation of normal human keratinocytes undergoing aging in vitro. Ann N Y Acad Sci. 2006;1067:332–6.

23.

Shin

Choi

Nam

Lee

Kim

Joe

Kazumi

Park

KC.

The co-expression pattern of p63 and HDAC1: a potential way to disclose stem cells in interfollicular epidermis. Int J Mol Sci. 2017;18:1360.

24.

Kim

Villadsen

Expression of luminal progenitor marker CD117 in the human breast gland. J Histochem Cytochem. 2018;66:879–88.

25.

Garbe

Pepin

Pelissier

Sputova

Fridriksdottir

Guo

Villadsen

Park

Petersen

Borowsky

Stampfer

LaBarge

MA.

Accumulation of multipotent progenitors with a basal differentiation bias during aging of human mammary epithelia. Cancer Res. 2012;72:3687–701.

26.

Lim

Pal

Bouras

Asselin-Labat

M-L

Vaillant

Yagita

Lindeman

Smyth

Visvader

JE.

Transcriptome analyses of mouse and human mammary cell subpopulations reveal multiple conserved genes and pathways. Breast Cancer Res. 2010;12:R21.

27.

Györffy

Lanczky

Eklund

Denkert

Budczies

Szallasi

An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast Cancer Res Treat. 2010;123:725–31.

28.

Paramio

Casanova

Segrelles

Mittnacht

Lane

Jorcano

JL.

Modulation of cell proliferation by cytokeratins K10 and K16. Mol Cell Biol. 1999;19:3086–94.

29.

Santos

Paramio

JSM

Bravo

Ramirez

Jorcano

JL.

The expression of keratin k10 in the basal layer of the epidermis inhibits cell proliferation and prevents skin tumorigenesis. J Biol Chem. 2002;277:19122–30.

30.

Petersen

Van Deurs

Growth factor control of myoepithelial-cell differentiation in cultures of human mammary gland. Differentiation. 1988;39:197–215.

31.

Habif

Perzin

Lipton

Lattes

Subareolar abscess associated with squamous metaplasia of lactiferous ducts. Am J Surg. 1970;119:523–26.

32.

Hurt

Díaz-Arias

Rosenholtz

Havey

Stephenson

HE.

Posttraumatic lobular squamous metaplasia of breast. An unusual pseudocarcinomatous metaplasia resembling squamous (necrotizing) sialometaplasia of the salivary gland. Mod Pathol. 1988;1:385–90.

33.

Santini

Ceccarelli

Taffurelli

Pileri

Marrano

Differentiation pathways in primary invasive breast carcinoma as suggested by intermediate filament and biopathological marker expression. J Pathol. 1996;179:386–91.

34.

Bratthauer

Miettinen

Tavassoli

FA.

Cytokeratin immunoreactivity in lobular intraepithelial neoplasia. J Histochem Cytochem. 2003;51:1527–31.

35.

Kim

Villadsen

Sorlie

Fogh

Gronlund

Fridriksdottir

Kuhn

Rank

Wielenga

Solvang

Edwards

PAW

Borresen-Dale

A-L

Ronnov-Jessen

Bissell

Petersen

OW.

Tumor initiating but differentiated luminal-like breast cancer cells are highly invasive in the absence of basal-like activity. Proc Natl Acad Sci U S A. 2012;109:6124–9.

36.

Bagger

Hopkinson

Pandey

Bak

Brydholm

Villadsen

Helin

Rønnov-Jessen

Petersen

Kim

Aggressiveness of non-EMT breast cancer cells relies on FBXO11 activity. Mol Cancer. 2018;17:171.

37.

Xin-RongYang Xu

Shi

Fan

Zhou

Sun

Qiu

Gao

Qin

Chen

Yang

Tang

ZY.

Cytokeratin 10 and cytokeratin 19: predictive markers for poor prognosis in hepatocellular carcinoma patients after curative resection. Clin Cancer Res. 2008;14:3850–9.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

1.62 MB

The Expression Pattern of Epidermal Differentiation Marker Keratin 10 in the Normal Human Breast and Breast Cancer Cells

Abstract

Keywords

Introduction

Materials and Methods

Human Breast Tissue Samples

Immunostaining

Microscopy and Image Acquisition

Cell Culture

RNA Isolation and Real-time Quantitative PCR

Kaplan–Meier Plots

Results

Rare K10pos Luminal Cells Are Most Frequently Present in Breast Gland Structures of Women >30 Years of Age

K10pos Cells Belong to a Mature Luminal Compartment

K10pos Breast Carcinomas Mimic a Normal Luminal Differentiation Pattern

Expression of KRT10 Correlates to a Worse Outcome for Breast Cancer Patients

Discussion

Supplemental Material

2019-00222R1_Production_Supplemental_Material_online_supp – Supplemental material for The Expression Pattern of Epidermal Differentiation Marker Keratin 10 in the Normal Human Breast and Breast Cancer Cells

Footnotes

Acknowledgements

Competing Interests

Author Contributions

Funding

References

Supplementary Material

Rare K10^pos Luminal Cells Are Most Frequently Present in Breast Gland Structures of Women >30 Years of Age

K10^pos Cells Belong to a Mature Luminal Compartment

K10^pos Breast Carcinomas Mimic a Normal Luminal Differentiation Pattern