Nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS) is 27 kDa chromosomal protein of unknown function. Its amino acid composition as well as structure of its DNA binding domain resembles that of high-mobility group A, HMGA proteins. HMGA proteins are associated with various malignancies. Since changes in expression of HMGA are considered as marker of tumor progression, it is possible that similar changes in expression of NUCKS could be useful tool in diagnosis and prognosis of breast cancer. For identification and analysis of NUCKS we used proteomic and histochemical methods. Analysis of patient-matched samples of normal and breast cancer by mass spectrometry revealed elevated levels of NUCKS in protein extracts from ductal breast cancers. We elicited specific antibodies against NUCKS and used them for immunohistochemistry in invasive ductal carcinoma of breast. We found high expression of NUCKS in 84.3% of cancer cells. We suggest that such overexpression of NUCKS can play significant role in breast cancer biology.
Nuclear ubiquitous casein and cyclin-dependent kinases substrate (NUCKS) is a nuclear DNA binding protein occurring in almost all types of human cells, adult and fetal tissues [
The benign human tumors are mainly of mesenchymal origin and result from chromosomal rearrangements. These include lipomas [
The abundance of NUCKS in rapidly growing cells as well as the overexpression of
Samples of IDC of grades II or III were retrieved during surgery. Analysis of the samples followed an informed consent approved by the local ethics committee. The entire protein extraction procedure was carried out as described previously [
Aliquots of protein fractions containing NUCKS were separated by SDS-PAGE, using NuPAGE Novex Bis-Tris 4%–12% gels (Invitrogen, Carlsbad, CA, USA) and MES running buffer according to the manufacturer's instructions. The gel was stained with Coomassie Blue using Colloidal Blue Staining Kit (Invitrogen).
The NUCKS bands were subjected to a standard in-gel trypsin digestion protocol [
Peptide mixtures were separated by online reversed-phase nanoscale capillary liquid chromatography and analyzed by electrospray tandem mass spectrometry as described previously [
Antibodies against NUCKS were elicited in rabbits using synthetic peptide DEDYGRDSGPPTKKC (residues 23–26) conjugated to ovalbumin (Imject Maleimide Activated Ovalbumin, Pierce). Animals were injected with 0.2 mg of cross-linked peptide. Titer and specificity of the antisera were increased by three injections (boosts).
Monospecific antibodies were purified on affinity columns which were prepared by coupling of the peptide to iodoacetate activated gel (SulfoLink, Pierce) according to the manufacturer’s protocol using 1 mg of the peptide per 1 mL of the coupling gel. The unreacted maleimide groups were quenched with 20 mM cysteine. Then 1 mL of the gel was loaded into a column and washed with 50 mL PBS. 5 mL of the antiserum was diluted with 5 mL PBS and incubated with the gel for 4 hour. Following washing of the gel with 50 mL PBS the bound antibodies were eluted with 10 mL of 0.1 M glycine-HCl, pH 2.5, and after that with 10 mL 4 M guanidine hydrochloride in PBS. The eluates were diluted with 10 mL of PBS and concentrated in Centriprep-Ultracel YM-10 (Millipore) concentrators to a volume of 2 mL. The concentrates were dialyzed against PBS overnight. The guanidine hydrochloride fraction of antibodies was used in all experiments.
For Western analysis, proteins were transferred from SDS gels onto the nitrocellulose membrane by electroblotting at 10 V/cm for 40 min. The proteins were cross-linked to the membrane by incubation in 0.5% (v/v) glutaraldehyde in PBS for 10 min. The membranes were blocked with 10% (v/v) normal goat serum (NGS) for 30 min prior to incubation with the primary antibodies together with 1% NGS in PBS containing 0.1% Tween 20 (PBS-T). The concentration of the primary antibodies was 1.5
26 samples of breast cancer, derived from 26 different female patients, were investigated in this experiment. In particular, this was the invasive ductal carcinoma (IDC) of grade (G) I (10 cases), II (6 cases), and III (10 cases). Cancer was excised during radical mastectomy or tumorectomy depending on the result of the previously performed imaging studies, fine-needle aspiration biopsy, oligobiopsy, or intraoperative diagnosis.
The samples from the tumors were fixed in 4% formalin and embedded in paraffin. After that, the paraffin blocks were cut on microtome to obtain 4
Following deparaffinization the antigen determinant was retrieved using normal pressure cooking in 0.01 M sodium citrate for 9 minutes in 350 W microwave oven at pH 6. The slides were blocked for endogenous peroxidase (Peroxidase Blocking Reagent, DAKO Cytomation) and incubated overnight with anti-NUCKS antibodies at concentration of 15
Percentage of positively stained cancer cells was evaluated on 5 fields from the centre of tumor: number of positively stained cells (nuclei and cytoplasm) was divided by the total number of cancer cells seen on that field at magnification of 400 times. A mean value from 5 such fields is shown in “results” section. The selection criterion for the 5 fields measured was the centre of each sample and this was assessed by two pathologists. We also evaluated staining in other cells: lymphocytes and endothelial cells used as positive control because it was suggested that NUCKS is localized in both proliferating and nonproliferating cells [
The microphotographs were taken using light microscope (OLYMPUS BX40) at magnification 200 times and digital camera DP10. Two independent pathologists evaluated each slide. Statistical analysis of correlation between histological grading and number of positively stained cells was made. ANOVA one-way analysis of variance by ranks was used for this statistical analysis.
A group of nuclear proteins including linker histones H1 and HMG proteins can be selectively extracted from cells and tissues with diluted perchloric acid [
We found that the band of NUCKS with a relative Mr of 43,000 occurred abundantly in all samples of IDC, whereas in all normal samples either it did not appear or the band was very weak (Figure
NUCKS is highly overexpressed in breast cancer. Lanes 2–11: Protein extracts from five pairs of tissue, each pair matching the individual patient, of normal breast (N) and invasive ductal carcinoma GII (Ca) were separated by SDS-PAGE and stained with Coomassie.
To investigate the occurrence of NUCKS in cells and tissues, antibodies against a peptide corresponding to residues 23–36 of the protein were elicited in rabbits and were affinity purified. The specificity of the antibodies was analyzed on western blots using purified NUCKS, perchloric acid extracts and whole SDS whole lysates of MCF7 cells, and IDC and normal breast tissue (Figure
Specificity of the antibodies elicited against NUCKS’ peptide: Purified NUCKS ((a) lanes 2–4), HClO4-extracts invasive ductal carcinoma GII (Ca in (b): lanes 5 and 7) and MCF 7 cells ((b): lanes 6 and 8), and SDS-total lysates of normal breast (N: lanes 9, 10, 13, 14) and invasive ductal carcinoma GII (Ca in (c): lanes 11, 12, 15, 16) each matching the same patient (c) were separated by SDS-PAGE, blotted onto nitrocellulose and incubated with affinity purified anti-NUCKS antibodies. Lane 4: the antibodies were preincubated with the peptide used for antibody production and purification. Total protein was stained on blots with Amido Black (lanes 1, 2, 5, 6, and 9–12).
Purified NUCKS
HClO4 extracts
SDS total lystates
The study comprised 26 cases of invasive ductal carcinoma (IDC). They were first evaluated with regard to histopathology (grading) and then the samples from particular tumors were studied using immunohistochemical method. The occurrence of NUCKS was observed in tumor cells as well as in other cells seen in samples, like endothelia and lymphocytes.
In invasive ductal carcinoma, IDC (
The NUCKS positive staining in cells of invasive ductal carcinoma (in %) with relation to histological grading (I-II-III).
Grade | Number of cases | % stained cell (SD) |
---|---|---|
I | 10 | 77.5 (5.36) |
II | 6 | 85.5 (4.59) |
III | 10 | 89.6 (3.47) |
Total | 26 | 84.3 |
Occurrence of NUCKS in invasive ductal carcinoma, G III. The positive reaction was found in majority of tumor cells with prevalence of nuclear staining (arrows and brown color). The nuclei negative for NUCKS are stained blue or dark blue. ABC method. Magnification 200x, inset 40x.
Occurrence of NUCKS in invasive ductal carcinoma, GII. The positive staining in this case was less frequent than that in the case shown in Figure
Occurrence of NUCKS in invasive ductal carcinoma, GI. The positive staining in this case was the least frequent in comparison to cases of IDC GII and GIII. ABC method. Magnification 200x, inset 40x.
Positive control for NUCKS. This was obtained using positive reaction for NUCKS in lymphocytes from an inflammatory infiltration at the border of invasive ductal carcinoma. ABC method. Magnification 200x.
Negative control. This was obtained by omitting the primary antibody. The sample is from invasive ductal carcinoma, GII. Sample is counterstained with HE. Magnification 200x.
Normal breast. This figure presents the fat tissue from the IDC patient at a morphologically normal site away from the lesions which showed no reaction to anti-NUCKS antibody. ABC method, Magnification 200x.
Considering the significance level,
Comparison of
GI | versus | GII | .002352 |
GI | versus | GIII | .000004 |
GII | versus | GIII | .093116 |
Posthoc test results of the least significant differences, LSD, between different groups of cancer with regard to grading (axis X-groups, axis Y-fractions of stained cells).
Until now NUCKS was studied in detail using a variety of biochemical and cell biological methods [
NUCKS has 2 regions termed as nuclear localization signals (NLSs) where NLS1 is assumed to be main nuclear localization signal that binds to importin
Grade III tumors were found to manifest high levels of several genes involved in regulation of gene expression including NUCKS [
The NUCKS was reported to localize in both proliferating and nonproliferating cells: positive staining for NUCKS was found in our study in endothelial cells and lymphocytes. Since endothelial cells and lymphocytes belong to the relatively fast growing cells or at least they show high metabolism, overexpression of NUCKS is probably related to high levels of transcription.
In our study the positive staining was mainly observed in nuclei of tumor cells and nuclei of normal cells. Mixed nuclear and cytoplasmic reaction, which was seen in cells of IDC, may reflect known distribution of NUCKS throughout the cytoplasm in mitotic cells whereas nuclear localization of NUCKS is connected with telophase of cellular cycle [
Previously, the expression of the high-mobility group protein gene
Recently, the elevated expression of DYRK3, NUCKS, COX-2, and translin and tubulin-
In conclusion, it has to be emphasized that further studies are needed to determine a role of NUCKS in human cancer and that this work provides only early insights in the abundant occurrence of the protein in breast cancer. The future work also should pay additional attention in elucidating potential functions of specific posttranslational modifications of NUCKS [
The authors thank Dr. M. Mann for continuous support and interest. The work was funded by: Wrocław Medical University, Poland, and Max-Planck Society.