ONCOLOGY LETTERS 18: 2853-2860, 2019 Characterization of Kelch domain‑containing protein 7B in breast tumours and breast cancer cell lines ANA MARTÍN-PARDILLOS1,2 and SANTIAGO RAMÓN Y CAJAL1‑3 1 Translational Molecular Pathology Group, Oncology Department, Vall d'Hebron Institut de Recerca, Edificio Collserola, Hospital Vall d'Hebron, 08035 Barcelona; 2Centro de Investigación Biomédica en Red de Cáncer, 28029 Madrid; 3 Hospital Vall d'Hebron, Anatomical Pathology Department, 08035 Barcelona, Spain Received December 23, 2018; Accepted May 29, 2019 DOI: 10.3892/ol.2019.10672 Abstract. Adenocarcinomas exhibit great heterogeneity, line MCF‑10A and in the breast cancer cell lines MCF‑7, with many genetic and epigenetic alterations. The Kelch MDA‑MB‑231 and MDA‑MB‑468, using qPCR and western domain‑containing protein 7B (KLHDC7B) has recently blotting. The results revealed that all tested cancer cell lines been identified as epigenetically modified and upregulated in overexpressed KLHDC7B mRNA, but MDA‑MB‑468 exhib- breast cancer. The potential reversibility of epigenetic states ited a much lower level of protein expression relative to mRNA. offers exciting possibilities for novel cancer diagnostics and Although the breast cancer cell lines used may be appropriate drugs. However, to properly evaluate specific inhibitors, the for studying KLHDC7B epigenetic status, MDA‑MB‑468 role of KLHDC7B in the development and progression of should be excluded from functional experiments. breast cancer should be established. With that objective in mind, the present study investigated a series of human breast Introduction tumours and correlated their clinicopathology, according to the Elston‑Ellis modification of the Scarff‑Bloom‑Richardson Breast cancer is the second most common cancer worldwide and (SBR) grading system, with KLHDC7B mRNA expression, the most frequent cancer among women (1). One in 8 women in analysed using quantitative PCR (qPCR). The results revealed Europe will develop breast cancer before the age of 85 (2). The that KLHDC7B was significantly upregulated in grade 3 highest prevalence is found in northern and western European tumours, and that KLHDC7B expression varied according countries, suggesting a relation to environmental factors (3,4). to the tumour grade and the individual, being downregulated Metastatic breast cancer is the second leading cause of in well‑differentiated and moderately‑differentiated tumours cancer‑related fatality in women: It has a five‑year relative (grade 1‑2) and upregulated in poorly‑differentiated tumours survival rate of 23%, compared with 99% for non‑metastatic (grade 3). Immunohistochemical staining revealed that ductal breast cancer (5). Breast tumours are classified according to tumours and tumours with a higher percentage of Ki67 positive the Elston‑Ellis modification of the Scarff‑Bloom‑Richardson cells showed the highest levels of KLHDC7B. Receptor expres- (SBR) grading system (also known as the Nottingham grading sion, HER, p53 status, presence of metastasis, and vascular system) (6). This system grades tumours according to their invasion showed no association with KLHDC7B expression. differentiation, from well differentiated (grade 1), to moderately Previous studies have proposed KLHDC7B as an epigenetic differentiated (grade 2) or poorly differentiated (grade 3). This marker of breast cancer. We propose that KLHDC7B should be histological scale is used as a prognostic predictor in patients used as a marker for poorly‑differentiated tumours only; use of with breast cancer, as tumour grade has a positive correlation KLHDC7B without considering tumour grade could lead to an with metastasis and risk of recurrence. inaccurate diagnosis. Finally, we suggest the appropriate breast Regulation of signal transduction and stress response cancer cell lines to use to determine the functions of KLHDC7B. is critical to maintain cellular homeostasis. Cell signals in KLHDC7B expression was tested in the non‑tumour cell response to stress can result in growth arrest and elimination of damaged or premalignant cells. During cancer development, signalling pathways are often impaired due to dysregulation of gene expression or aberrant signal transduction, resulting in the hallmarks of cancer (7‑13). Gene expression is largely Correspondence to: Dr Ana Martín Pardillos, Translational regulated by epigenetic changes, including DNA methyla- Molecular Pathology Group, Oncology Department, Vall d'Hebron Institut de Recerca, Edificio Collserola, Hospital Vall d'Hebron, tion and histone modification (14). More than 50% of human Passeig de la Vall d'Hebron 119‑129, 08035 Barcelona, Spain cancers harbour mutations in enzymes involved in chromatin organization (15). One of the biggest challenges in cancer Key words: Kelch domain‑containing protein 7B, epigenetic, research is to understand how defects arise during disease breast, cancer, methylation, biomarker progression. This is likely to become increasingly important to detect and validate biomarkers for tumour gradings and subtypes that may help guide treatment decisions (16). 2854 MARTÍN-PARDILLOS and RAMÓN Y CAJAL: CHARACTERIZATION OF KLHDC7B IN BREAST CANCER One gene that has recently been found to be involved in (Barcelona, Spain). The study was approved by the Clinical breast and ovarian cancer and lymph node metastasis in Research Ethics Committee at Vall d'Hebron Hospital cervical cancer is the Kelch domain‑containing protein 7B [PR(AG)309/2016], and written informed consent was (KLHDC7B) gene (13,17‑19). It has been identified as hyper- obtained from patients prior to sample collection. Tissues were methylated and upregulated in breast cancer (17), and, when extracted during 2009 and mRNA was extracted from 2015 associated with alternative splicing events, may be involved to 2017. The selection criteria allowed different tumour types in the development and progression of cervical squamous cell (papillary, ductal, lobular, mucinous, tubular and ductal) and carcinoma (CSCC) (20). grades, including metastatic and non‑metastatic tumours. The KLHDC7B gene (Hs.137007) (21) comprises a single Tumours were classified according to the Elston‑Ellis exon, located on human chromosome 22q13.33 (22,23). In 50 modification of the Scarff‑Bloom‑Richardson (SBR) grading paired samples of breast cancer tissue and adjacent normal system (Nottingham grading system) (6) as well differentiated tissue, the methylation level of the 14 CpG sites at the promoter (grade 1, n=5), moderately differentiated (grade 2, n=10) or region of the gene was higher in cancerous tissue (72‑93%) than poorly differentiated (grade 3, n=11). in normal tissue (31‑83%) (17). A clear relationship between high methylation levels and upregulated expression was also Histology and immunohistochemistry. Immunohistochemical observed in cultured breast cell lines. For instance, MCF‑7 staining were performed on five‑micron‑thick sections from (90‑100%) and MDA‑MB‑468 (100%) cancer cell lines had formalin fixed and paraffin embedded (FFPE) tissues, on the higher methylation at the 14 CpGs and higher gene expression Ventana Benchmark XT Automated IHC Stainer, using the than BT549 (20‑90%) and 184B5 (10‑100%) cell lines (17). Ventana ultraView Universal DAB Detection kit (760‑500). Numerous reports have described an association between After deparaffinization with Ventana EZ Prep solution hypermethylation of individual genes and clinical prognosis (950‑102), antigen retrieval was performed using Ventana for various types of cancer, and individual methylation markers Tris‑based buffer solution CC1 pH 8 (950‑124). Endogenous have previously been linked to breast cancer metastasis (24). peroxidase was blocked with 3% hydrogen peroxide. After DNA methylation is generally associated with gene down- rinsing using Reaction Buffer (950‑300), slides were incubated regulation. However, some genes, including survivin (25), the at 37˚C with each primary antibody (Ventana Medical Systems glycoprotein hormone alpha‑subunit (26), and KLHDC7B, Inc, Tucson, AZ, USA; EEUU): Ki67‑20 min (rabbit mono- have been found to be upregulated when CpG sites are hyper- clonal antibody, 790‑4286), p53‑44 min (mouse monoclonal, methylated (17). 800‑2912), HER2/neu‑28 min (rabbit monoclonal, 790‑2991), The potential reversibility of epigenetic status offers ER‑40 min (rabbit monoclonal, 790‑4324) and PR‑16 min exciting opportunities for cancer treatments, and targeting (rabbit monoclonal, 790‑2223). Following incubation with HRP methylation represents the third wave of anticancer drug Multimer secondary antibody, primary antibodies‑horseradish development (24). DNA methyltransferases currently represent peroxidase‑labelled antibody complex were visualized using one of the major drug targets, and new drugs are expected to diaminobenzidine tetrahydrochloride chromogen. Slides were be added in the near future (24,27,28). then counterstained for 8 min with haematoxylin (760‑2021), The KLHDC7B gene encodes a 594‑amino‑acid protein for 4 min with bluing reagent (760‑2037), dehydrated and product that contains a Kelch domain in the C‑terminal mounted. Appropriate positive and negative controls were half (29). The Kelch domain is a common motif that forms a included within the study sections. 4‑stranded anti‑parallel β‑propeller. Kelch‑repeat β‑propellers interact with a variety of other proteins (30,31). Besides the Cell culture and reagents. MCF‑10A, MCF‑7, MDA‑MB‑231 presence of the Kelch domain and a verified expressed sequence and MDA‑MB‑468 breast cancer cell lines were purchased tag (EST), no other information is available to determine the from the American Type Culture Collection (ATCC, function of KLHDC7B (22). Kelch motif‑containing proteins Manassas, VA, USA) and authenticated by DNA profiling are involved in diverse biological processes, such as signal using short tandem repeat (STR) (GenePrint ® 10 System, transduction, building cell structures, regulating transcrip- Promega, Fitchburg, WI, USA) at Genomics Core Facility, tion, metabolism and, notably, in stress responses (22,32,33). Instituto de Investigaciones Biomédicas ‘Alberto Sols’ Mutations of Kelch proteins have been associated with cancer: CSIC‑UAM (Madrid, Spain) (35). Mycoplasma PCR analysis Examples include KLHL6 in lymphocytic leukaemia, KEAP1 detected no genetic material. Cells were maintained at 37˚C in pulmonary papillary adenocarcinoma, KLHL20 in prostate in a 5% CO 2 humidified incubator (AutoFlow UN‑5510, cancer, and KLHL37 (ENC1) in brain tumours (34). A recent Nuaire, Plymouth, MN, USA). MCF‑7, MDA‑MB‑231 and study showed that apoptosis of MCF‑7 decreased and prolif- MDA‑MB‑468 cells were cultured in Dulbecco's modified eration increased when KLHDC7B was upregulated, and Eagle's medium (DMEM) (Life Technologies, Carlsbad, when KLHDC7B was downregulated, the opposite occurred, CA, USA) supplemented with 10% heat‑inactivated fetal indicating its oncogenic properties (19). However, the encoded bovine serum (FBS; Biowest, Nuaillé, France) and antibiotics protein and its role in these cancers remain largely unknown. (penicillin, streptomycin; Gibco‑ThermoFisher Scientific, Waltham, MA, USA). MCF‑10A medium was additionally Materials and methods supplemented with 20 ng/ml EGF (cat. no: E9644; Sigma, St. Louis, MO, USA), 0.5 µg/ml hydrocortisone, 100 ng/ml Tumour samples. Breast cancer specimens (n=26) and adja- cholera toxin (cat. no: C9903; Sigma) and 10 µg/ml insulin cent healthy tissue specimens (n=17) were obtained from (cat. no: I9278; Sigma). Cells were trypsinized and passaged female patients with breast cancer at Vall d'Hebron Hospital using TrypLE reagent (ThermoFisher Scientific). ONCOLOGY LETTERS 18: 2853-2860, 2019 2855 Table I. Cell line characterization: Oestrogen receptor, progesterone receptor and p53 status, tumour subtype, origin and morphology. Cell line ER PR HER2 p53 Subtype Origin Morphology MCF‑10A ‑ ‑ ‑ Wild type Non‑tumour Fibrocystic disease Epithelial MCF‑7 + + ‑ Wild type Luminal A Adenocarcinoma Most differentiated; tight cell‑cell junctions MDA‑MB‑468 ‑ ‑ ‑ Mutated Triple negative A Adenocarcinoma Core basal‑like MDA‑MB‑231 ‑ ‑ ‑ Mutated Triple negative B Adenocarcinoma Least differentiated and highly invasive ER, oestrogen receptor; PR, progesterone receptor; HER2, human epidermal growth factor receptor 2. MCF‑10A is a non‑tumour breast cell line, which is The average KLHDC7B expression for grade 1 (G1) hormone‑receptor [oestrogen‑receptor (ER) and proges- tumours (n=5) was calculated, and mRNA expression of every terone‑receptor (PR)] negative, HER2 negative and p53 tumour was reported as relative to this average (value=1). The wildtype (36‑38) (Table I). The three breast cancer cell lines two G1 tumours with the highest KLHDC7B expression were derive from breast adenocarcinomas (Table I). MCF‑7 is clas- used as cut‑offs for low expression (G1 tumours being those sified as luminal A molecular subtype, hormone‑receptor with the lowest KLHDC7B expression overall) (Table II). positive, HER2 negative and p53 wildtype. Luminal A cancers Comparison between healthy and tumour tissue was are low‑grade, tend to grow slowly and have the best prognosis. performed in cases in which there was enough mRNA from MDA‑MB‑231 and MDA‑MB‑468 are triple‑negative/basal‑like healthy tissue to perform retrotranscription from mRNA to breast cancer cell lines, hormone‑receptor negative, HER2 nega- cDNA. tive and p53 mutated. Although both the MDA‑MB cell lines are triple‑negative, they show significant differences: MDA‑MB‑468 Protein extraction and immunoblotting. Total protein extracts is classified as type A, showing a core basal‑like morphology, and were generated using a RIPA Lysis Buffer System (sc24948; MDA‑MB‑231 is classified as type B, being the least differenti- SantaCruz Biotechnology, Dallas, TX, USA) supplemented ated, highly invasive and having the worst prognosis (39,40). with Protease Inhibitor Cocktail set III (539134; Calbiochem, San Diego, CA, USA) and Phosphatase Inhibitor Cocktail Set RNA extraction and quantification. Tissue samples were lysed II (524625, Calbiochem). Protein was quantified using a BCA using Tissue Lyser II (Qiagen, Venlo, the Netherlands) and Protein Assay kit (23225; ThermoFisher Scientific). Protein RNA was extracted by the L'Hospital Universitari Vall d'Hebron extracts (15 µg per sample) were loaded onto SDS‑PAGE gels Biobank (HUVH Biobank, Barcelona, Spain), using QuickGene and electrophoretically transferred to polyvinylidene fluoride RNA tissue SII kit (RT‑S2) (Fujifilm, Neuss, Germany) in (PVDF) membranes. Membranes were blocked with 5% BSA the automated nucleic acid extraction system QuickGene 810 in TBS‑T (Tris‑buffered saline, 0.1% Tween 20). Membranes (Fujifilm), according to the manufacturer's instructions. RNA were incubated with KLHDC7B antibody (ab126063; from culture cell lines was extracted using the PureLink™ RNA Abcam, Cambridge, UK) diluted at a ratio of 1:500 according Mini Kit (ThermoFisher Scientific). Quantification and assess- to the manufacturer's instructions and incubated with the ment of RNA purity was performed using a NanoDrop ND2000 membranes overnight at 4˚C. Goat horseradish peroxidase Spectrophotometer (ThermoFisher Scientific) and confirmed (HRP)‑linked secondary antibody was added at a dilution according to the RIN (RNA integrity number) using an Agilent ratio of (1:5,000) (31460; Pierce ThermoScientific) and Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). incubated with the membranes at room temperature for 1 h. β ‑actin (JLA20; Calbiochem) was used as housekeeping RT‑PCR. One microgram of total RNA was used to synthesize (1:15,000, 1 h at room temperature, secondary antibody cDNA using Maxima Reverse Transcriptase (ThermoFisher not required). The membranes were washed 3 times with Scientific) on a Veriti 96‑well Thermal Cycler (Applied TBS‑T. Immunodetection of proteins was performed using Biosystems, ThermoFisher Scientific). RT‑qPCR was Amersham ECL Western Blotting Detection Reagent (GE performed according to the manufacturer's instructions, on an Healthcare, Chicago, IL, USA) according to the manufac- Applied Biosystems ABI 7500 Fast Real Time‑PCR sequence turer's instructions. detection system, using Taqman Technology (ThermoFisher Scientific): TaqMan GeX Master Mix (4369016), KLHDC7B Statistical analysis. Prism 5.0 software (GraphPad Software probe (Hs00536653_s1) and HPOL probe (Hs00172187_m1) Inc., La Jolla, CA, USA) was used for statistics and data as housekeeping. RNA from healthy tissue was used as a representation. Data are presented as the mean ± standard normalisation control: FirstChoice ® Human Breast Total error of the mean. Significant differences were determined RNA (AM6952, AppliedBiosystems‑Ambion‑Thermo Fisher). using ANOVA with Dunnett's multiple comparisons test. Analysis of relative gene expression data was conducted using P<0.05 was considered to indicate a statistically significant RT‑qPCR and the 2‑ΔΔCq method (41). difference. 2856 MARTÍN-PARDILLOS and RAMÓN Y CAJAL: CHARACTERIZATION OF KLHDC7B IN BREAST CANCER Table II. Tumour features. Tumour type Ki67 (% Relative mRNA (breast positive Vascular expression (to Patient ID carcinoma) Grade ER PR HER p53 cells) invasion Metastases G1 average=1) B09‑13526 Papillary 2 +++ +++ ‑ ‑ 5‑10 ‑ No 0.18 B09‑3889 Ductal 2 ? ? ? ? ? ‑ No 0.43 B09‑24083 Lobular 1 +++ +++ ‑ ‑ <5 ‑ Yes 0.58 B09‑16306 Mucinous 1 +++ +++ ‑ ‑ <1 ‑ No 0.60 B09‑12616 Cribriform/tubular 1 +++ +++ ‑ ? ? ‑ No 0.77 B09‑5308 Ductal 3 ? ? ? ? ? ‑ No 0.77 B09‑18339 Lobular 2 +++ + ‑ ‑ 15 ‑ No 0.87 B09‑11839 Ductal 2 +++ ‑ ‑ ‑ 5 ‑ Yes 0.91 B09‑18841 Ductal 3 +++ +++ ‑ ‑ 5 ‑ Yes 1.03 B09‑16005 Ductal 3 +++ +++ ‑ + 60 + Yes 1.09 B09‑988 Lobular 2 ? ? ? ? ? ? Yes 1.21 B09‑25265 Lobular 2 +++ +++ ‑ ‑ 2 ‑ Yes 1.21 B09‑19643 Lobular 2 ? ? ? ? ? ? No 1.34 B09‑24926a Ductal 1 +++ +++ ‑ ‑ <5 + Yes 1.48 B09‑24052 Ductal 3 +++ +++ ‑ ‑ 20 ‑ No 1.52 B09‑22493 Lobular 2 +++ +++ ‑ ‑ <5 ‑ No 1.63 B09‑24372a Ductal 1 +++ +++ ‑ ‑ 1 ‑ No 1.64 B09‑8511 Ductal 2 +++ +++ ‑ ‑ 15 ‑ Yes 1.69 B09‑7717 Ductal 3 +++ ++ ‑ + 70 ‑ ? 1.79 B09‑11451 Papillary 3 +++ ++ ‑ ? ? ‑ ? 1.80 B09‑3173 Ductal 3 ‑ ‑ ‑ + >70 + Yes 1.85 B09‑26055 Ductal 3 +++ + ‑ ‑ 40 ‑ Yes 1.98 B09‑1573 Tubular 2 ? ? ? ? ? ? Yes 2.11 B09‑20004 Ductal 2 +++ +++ ‑ ‑ 3 ‑ No 2.16 B09‑15644 Ductal 2 +++ ++ ‑ ‑ 15‑20 +/‑ Yes 2.26 B09‑20128 Papillary/Ductal 3 +++ +++ ‑ ‑ 10 ‑ No 2.43 B09‑20045 Ductal 3 ‑ ‑ ‑ + 60 ‑ No 3.02 B09‑25449 Ductal 2 ++ +++ ‑ ‑ 25 + Yes 3.70 B09‑2726 Ductal 3 ‑ ‑ ‑ + 50 + Yes 3.98 B09‑19151 Ductal 3 +++ +++ ‑ + 1 ‑ No 6.98 B09‑24264 Ductal 2 +++ +++ ‑ ‑ <5 ‑ No 23.44 B09‑17267 Ductal 3 ++ + + + 15‑20 + No 272.58 a The two G1 tumours with the highest KLHDC7B expression, used as the cut‑off for low expression. Average expression in G1 tumours (n=5) was calculated, and mRNA expression in each tumour was reported as relative to this average (value=1). ER, oestrogen receptor; PR, progesterone receptor; HER, human epidermal growth factor receptor; ?, unknown. Results tumour type showed a correlation with KLHDC7B expres- sion. Tumours with more than 10% Ki67 cells had the highest Clinicopathological evaluation and KLHDC7B characterization levels of KLHDC7B expression. For tumour type, lobular in breast cancer tumours. We studied the expression of KLHDC7B tumours had the lowest expression of KLHDC7B (83.33% mRNA in tumours from different pathological grades (Fig. 1A). of lobular tumours were classified as having low expression), Our results showed that KLHDC7B expression tended to increase and ductal tumours had the highest expression (68% of ductal as tumour grade increased from grade 1 to 2. Grade 3 tumours tumours were classified as having high expression). Papillary, showed a significant upregulation of KLHDC7B. mucinous, cribriform and tubular tumours were also analysed, Table II shows the classification of tumours in order of but due to the low number of each of these tumour types, we increasing KLHDC7B expression, with information on cannot draw conclusions on their relationship to KLHDC7B tumour type, tumour grade, hormone‑receptor (oestrogen and expression. Besides tumour type and percentage of posi- progesterone), HER and p53 status, percentage of Ki67 posi- tive Ki67 cells, no other correlations were found between tive cells, metastasis and vascular invasion. Ki67 cells and KLHDC7B and the tumour features described above. ONCOLOGY LETTERS 18: 2853-2860, 2019 2857 Figure 1. KLHDC7B mRNA expression in breast cancer. (A) mRNA expression in grade 1 (n=5), grade 2 (n=14) and grade 3 (n=9) breast tumours, relative to grade 1 tumours. (B) Relative mRNA expression in negative control (commercial RNA), non‑tumour surrounding breast tissue, and grade 1, 2 and 3 tumours. Expression relative to negative control. For each tumour grade, there is a corresponding mark of the same colour in non‑tumour tissue, allowing a comparison. Lines connect grade 3 tumours with their respective non‑tumour surrounding tissue (red, upregulated expression; green, downregulated expression). *P<0.05, as indicated. KLHDC7B, Kelch domain‑containing protein 7B. Figure 2. mRNA expression in breast tumours compared with non‑tumour surrounding tissue in the same patient. Expression relative to non‑tumour tissue. The size of the error bars reflects the variation in the replicates. KLHDC7B, Kelch domain‑containing protein 7B; Metastasis: N, No metastasis; M, Metastasis. In comparison to healthy tissue from the area surrounding to non‑tumour surrounding tissue. Only 3 out of 10 (30%) the tumour, with expression normalised to a commercial grade 2 tumours showed a significantly higher KLHDC7B RNA sample from a healthy donor (Fig. 1B), grade 3 tumours expression in tumour tissue. Four out of 5 (80%) grade 3 showed a tendency to KLHDC7B upregulation. However, the tumours showed a significantly higher KLHDC7B expression expression of KLHDC7B in grade 3 tumours was not always than healthy tissue. higher than non‑tumour samples from other patients (from When we correlated KLHDC7B expression with meta- grade 1‑ and 2‑matched tissue). static capacity (Fig. 2), 4 out of 10 (40%) of the tumours that To avoid the influence of tumour heterogeneity among produced metastases showed upregulation of this gene, but 5 patients, we compared the KLHDC7B expression in breast out of 10 (50%) had downregulation. tumours and in the surrounding healthy tissue in the same patient (Fig. 2). Seven out of 12 (58%) grade 1 and 2 tumours Characterization of KLHDC7B mRNA and protein expression showed a significantly lower KLHDC7B expression compared in cell lines. KLHDC7B mRNA expression was evaluated in 2858 MARTÍN-PARDILLOS and RAMÓN Y CAJAL: CHARACTERIZATION OF KLHDC7B IN BREAST CANCER Figure 3. KLHDC7B expression in breast cancer cell lines (MCF‑7, MDA‑MB‑231 and MDA‑MB‑468) compared with the non‑tumour cell line (MCF‑10A). (A) mRNA expression, relative to MCF‑10A. (B) Protein expression via western blotting, relative to MCF‑10A. (C) Representative image of protein expression as determined by western blotting. β‑actin was used as the internal control. *P<0.05, as indicated. KLHDC7B, Kelch domain‑containing protein 7B. different cell lines, using MCF‑10A as the non‑tumour cell approach confirmed previously published data on KLHDC7B lines and MCF‑7, MDA‑MB‑231 and MDA‑MB‑468 as the upregulation in breast tumours (17), but our results also revealed tumour cell lines. As expected, KLHDC7B was overexpressed that the expression of this gene is grade‑dependent and only in tumour cell lines (Fig. 3A). significantly upregulated in grade 3 tumours. Additionally, Protein expression was determined by western‑blot we found interesting results in grade 1 and 2 tumours, that (Fig. 3B and C). All four tumour cell lines showed higher KLHDC7B was downregulated in well‑differentiated and expression than MCF‑10A (no expression). Surprisingly, moderately‑differentiated tumours. These new data would MDA‑MB‑468 showed lower expression than the other tumour suggest a dual role of KLHDC7B during tumour progression, cell lines, although the mRNA expression was higher. which we will analyse in future studies. We can conclude that when using KLHDC7B expression as Discussion a marker of breast cancer, it should be correlated against healthy tissue from the same patient, rather than the general population, KLHDC7B has been found in different tumours including as comparisons with the general population are likely to lead breast, ovary and cervical cancer (13,17‑18,20), revealing its to false results (a consequence of intertumour heterogeneity). possible role in tumour biology. The KLHDC7B gene has been Additionally, use of KLHDC7B as a marker without consid- found to be hypermethylated and upregulated in breast cancer ering tumour grade could lead to inaccurate diagnoses. and consequently has been postulated as an epigenetic marker The results of this study could increase understanding of of breast cancer (17). However, its role in the development and the involvement of KLHDC7B in breast cancer, although the progression of these cancers is largely unknown. sample size poses a potential limitation, and future studies To understand the role of KLHDC7B in tumour progres- should use a larger sample. sion we studied the expression of KLHDC7B mRNA in These data indicate that KLHDC7B is associated with more tumours of different pathological grades. Grade 3 tumours, aggressive tumours and worse prognosis, however they do not tumours with more than 10% Ki67 positive cells and ductal explain the functional role of KLHDC7B in breast tumours. tumours had the highest expression of KLHDC7B (Table II). KLHDC7B protein could have an anti‑ or pro‑tumour role or Ki67 expression is a well‑known marker of active proliferation even a dual role that could explain the differences according and the association between high proliferation and poor prog- to tumour grade. To unravel the functional role of KLHDC7B, nosis is well stablished (42‑44). Lobular, mucinous, tubular, future experiments should be performed including up‑ and and papillary carcinomas have been associated with lower risk down‑regulation of KLHDC7B expression to establish its role of mortality than ductal carcinomas (45). Together, these data in the progression of breast tumours. These studies should indicate that KLHDC7B is associated with more aggressive preferably be performed in breast cell lines. tumours and worse prognosis. We studied the expression of KLHDC7B in tumour cell The upregulation of KLHDC7B in advanced tumours lines and healthy cell lines. MCF‑10A, MDA‑MB‑231 and could suggest a positive association with metastatic capacity, MDA‑MB‑468 are hormone‑receptor and HER negative cell although we did not find such a difference in our analysis. lines; MCF‑10A does not express KLHDC7B, while the two When we compared tumour tissue with healthy breast MDA‑MB cell lines do. In contrast, MCF‑7 is a hormone‑receptor tissue, KLHDC7B expression in tumour tissue was not always positive cell line and expresses KLHDC7B (Table I). Our higher than in non‑tumour samples from other patients with results therefore suggest that hormone‑receptor and HER grade 1 and 2 tumours. These data reveal a huge variability status are not related to KLHDC7B expression. among individuals, demonstrating one of the most relevant We have confirmed previously published results on mRNA issues in oncology‑intertumour heterogeneity. To improve expression and added protein expression studies, which have understanding of the role of KLHDC7B in breast cancer, not previously been described. Our data show that mRNA we compared KLHDC7B expression in breast tumours and expression does not correlate exactly with protein expression. surrounding healthy tissue from the same patient. 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