STYK1 expression in breast cancer and its association with vascular invasion and clinicopathological features

Abstract

Vascular invasion (VI) is an essential step in the progression of breast cancer (BC) and is a key factor behind morbidity and mortality. In a previous work using gene expression profiling, upregulation of STYK1 was one of the genetic changes that was found associated with VI. The aims of the following study were to assess (1) level and pattern of STYK1 expression in BC tissues and (2) its association with clinicopathological features and with patient outcome. Histopathological, clinical data and 10‒year survival data for 220 primary breast carcinoma was retrieved. From each specimen, one representative formalin-fixed paraffin-embedded tissue section was stained by immunohistochemistry (IHC) with STYK1 antibodies. IHC score was calculated and correlated with clinicopathological features of tumors. Higher STYK1 expression was detected in invasive BC compared with normal and non-invasive tumors, P = 0.001 and was significantly associated with the presence of vascular invasion (P = <0.001), lymph node metastasis (P = 0.032) estrogen receptor positivity (P = 0.033), non‒triple negative phenotype (P = <0.018) and occurrence of locoregional recurrence (P = 0.011). No association was detected with other tumor features such as tumor size, grade, development of distant metastasis or overall survival. STYK1 is expressed at a higher level in invasive BC compared with non-cancerous tissues. This high expression is significantly associated with presence of VI, LN metastasis and occurrence of recurrence. These findings suggest that STYK1 could be a possible molecular target for breast carcinoma. Targeted therapy that can inhibit STYK1 kinase can be a possible approach for the development of novel therapies in breast carcinoma.

Introduction

Vascular invasion is an essential step in the progression of any malignant tumor including breast cancer. Through VI, malignant cells spread to remote sites establishing new tumor deposits in distant organs causing tissue destruction and function deterioration which is the key factor behind morbidity and mortality of breast cancer. In previous work, we could identify genetic changes associated with the ability of breast cancer to invade tumor vasculature¹. Upregulation of STYK1 was one of the genetic alterations that came on top of these genetic changes. However, data about STYK1 expression in human breast cancer tissues or its association with clinicopathological features and clinical outcome is limited with some controversial findings among studies.

STYK1 is an oncogenic protein that belongs to the receptor tyrosine kinase (RTK) family. Members of this family are cell surface receptors. These receptors become activated when they bind the relevant growth factors, cytokines or hormones². Among the well-known groups of this family are the epidermal growth factor (EGF) proteins and platelet-derived growth factor/fibroblast growth factor (PDGF/FGF) receptor superfamilies. It was interesting to find that STYK1 shares approximately 30% amino acid identity with PDGF/FGF protein³.

Expression of STYK1 was examined in several tumor types. In lung carcinoma, STYK1 was detected at higher levels in tumor tissues compared to normal tissue and to be associated with higher proliferation, more advanced tumor stage and with poorer prognosis⁴. The same observation was detected with urinary bladder carcinoma⁵ and with gall bladder carcinoma⁶. In colorectal carcinoma, high STYK1 expression was associated with bigger tumor sizes but not with clinicopathological characteristics⁷. An opposite observation was reported with gastric carcinoma where downregulated STYK1 expression correlated significantly with poor tumor differentiation and with advanced clinical stage⁸.

In breast carcinoma, data about STYK1 expression in human tissues and its association with clinicopathological features is not sufficient particularly regarding association with vascular invasion. Few research studies assessed STYK1 in breast cancer could be retrieved from literature. In one study STYK1 expression was found in early stages in breast carcinoma and was suggested to be used as a diagnostic marker for malignancy⁹, in another study, STYK1 expression at level of mRNA was measured by quantitative polymerase chain reaction (QRT ‒PCR) in breast cancer cell line after estrogen treatment¹⁰. The findings from the mentioned study suggested that STYK1 function is affected by estrogen, however not fully known how and what will be the final effects. In an interesting recent work, STYK1 was found to increase breast cancer proliferation through synergistic action with the epidermal growth factor receptor¹¹.

Aims of this study were to assess (1) level and pattern of STYK1 protein expression in human breast cancer tissues from primary breast cancer (2) association between STYK1 expression and HER-2 expression to find if there is combined effect between these two molecules and if this has any implication to tumor progression and patient prognosis (3) association between STYK1 expression with clinicopathological features and with patient outcome.

Materials and methods

Specimens

In this study, histopathological and clinical data for 220 primary invasive breast carcinoma was retrieved. One representative formalin-fixed paraffin-embedded block that has enough tumor tissues and peritumoral area was selected for IHC staining and analysis for STYK1 (180 specimens). All specimens were either mastectomy or local excision biopsies. Histopathological types of breast carcinoma enrolled in this research study were as follows: 182 (82%) carcinomas of no specific type (NST), 23 (10%) lobular, 3 (1.3%) medullary, 2 (0.9%) metaplastic, 5 (2.2%) mucinous, 1(0.5) tubulolobuar, 1 (0.5) papillary and 5 (2.3%) mixed carcinomas. The histopathological grades of specimens were 16% grade I, 54% grade II and 30% grade III. Patients were older than 50 years in 52% of cases. Tumor size was larger than 2 cm in 146 (67%) specimens. Estrogen receptor (ER) status was positive in 173 (78%) specimens while progesterone receptors (PR) status was positive in 143 (66%). The cases studied were of stage II in (48%) and of stage III in (35%) of specimens. More details are mentioned in Table 1.

Table 1.

Clinicopathological features of specimens of the study.

Feature	Frequency (%)	Feature	Frequency (%)
Patient Age		ER status
≤ 50 Y	107 (48)	Negative	46 (22)
> 50 Y	113 (52)	Positive	173 (78)
No data	0	No data	1
Tumor size		PR status
≤ 2 cm	71 (33)	Negative	72 (34)
> 2 cm	146 (67)	Positive	143 (66)
No data	3	No data	5
LN status		Triple negative status
Negative	79 (36)	Non-TN	170 (87)
Positive	141 (64)	TN	25 (13)
No data	0	No data	25
Tumor grade		Tumor stage
I	34 (16)	I	39 (17)
II	117 (54)	II	104 (48)
III	65 (30)	III	77 (35)
No data	4	No data	0

Note: ER=estrogen receptor, PR=progesterone receptor, HER2 = human epidermal growth factor receptor-2, TN=triple-negative breast cancer, LN status= lymph node status.

This study utilized archived human tissue samples in the form of paraffin-embedded tissue blocks obtained from the pathology department’s repository at Salmaniya Medical Complex (SMC), Bahrain. These tissue blocks were originally collected for diagnostic purposes. Ethical approval for the use of these archived samples was granted by the Research and Ethics Committee at SMC (number 60-090524) as well as by the Research Ethics Committee of the Arabian Gulf University (number E6-PI-10-23). Both committees adhere to ethical guidelines that permit research on old, stored tissue samples without requiring individual patient consent, provided the samples are anonymized. This approach aligns with international ethical standards for retrospective studies involving archived human tissues.

Immunohistochemical staining

One 4 μm ‒ thickness tissue section was cut from each specimen and stained immunohistochemically with antibody against STYK1. First, sections were incubated in 55 °C oven for 15 min then were deparaffinized in two xylene changes 10 min each. Rehydration was done in descending concentration of ethanol Alcohol followed by immersion in distilled water (DW). For antigen retrieval, sections were put in citrate buffer (pH 6) then in microwave for 20 min. Reaction was left to cool at room temperature (RT) for about 30 min, then washed in DW for 2 min. Neutralize Endogenous peroxidase using Hydrogen peroxidase block then sections were rinsed with phosphate buffered saline (PBS), PH 7.6, for two times, 2 min each. STYK1 antibody was added to each section (Thermo Fisher Scientific Monoclonal (2H2F10) AB MA5 ‒15372 ) for 1 h at RT at 1:200 concentration. After incubation, excess Ab was washed with PBS twice, 5 min each. For visualization of the antibody reaction, Leica, Novolink™ Polymer Detection System kit, Product No: RE7280-K was used according to manufacturer instructions. Sections were washed in DW, counter stained with Mayer’s hematoxylin, washed again in DW, dehydrated in ascending grades of ethanol alcohol then cleared in xylene and finally mounted with DPX and cover slipped. Tissue sections from the placenta were used as a positive control. STYK1 is expressed in the trophoblastic layer. For negative control, section from the same tissue was used in each staining run after omission of the primary antibody.

Assessment of STYK1 expression

Positive STYK1 expression appeared as brown staining in the cytoplasm of the epithelial cells. The same as reported in previous studies^12,13. For quantification of STYK1 expression, staining intensity was graded into: 0 (complete negativity), 1 (weak staining intensity), 2 (moderate staining intensity) and 3 (strong staining intensity). Then, the percentage of positivity was multiplied by grade of intensity of the positive staining to give an immunohistochemistry score (IHC score) for each specimen. IHC scores ranged from 0 to 300. The median was used as a cutoff point according to which specimens were further categorized into STYK1‒ low expressing specimens and STYK1‒ high expressing specimens. Scoring was done manually by expert pathologists using the light microscope. For confirmation of scoring accuracy, image J analysis software (ImageJ 1.46r) was used to assess staining intensity in 20% of slides independently by another investigator blinded to the IHC scores. The digital quantification by ImageJ was done by calculating the percentage of positively stained area. The immunohistochemicaly-stained slides was first scanned by the virtual microscope, Aerio LV1 IVD live view and desktop scanner, Leica Biosystems imaging, Inc. High-resolution images were captured from representative tumor regions at ×200 magnification. The DAB chromogen signal was isolated using the color deconvolution plugin, and an intensity threshold was applied to identify positive staining. Three non-overlapping regions of interest (ROIs) were selected per slide, and the percentage of stained area within each ROI was measured. The average percentage across the ROIs was calculated for each case and used to categorize specimens as STYK1-low or STYK1-high. Analysis was performed by an independent investigator blinded to the manual IHC scores. All specimens that were categorized as STYK1-low or STYK1- high using the manual scoring was found to be of the same category using the ImageJ analysis giving a 100% concordance between both methods supporting the reliability and reproducibility of the manual scoring method.

STYK1 expression was examined in the invasive tumor tissue. When the invasive tumor has normal mammary tissue and/or carcinoma In situ tissues in the surrounding peritumoral area, scoring of STYK1 expression in these areas was done independently from the invasive tumor tissues.

For statistical analysis, 2 × 2 table and chi squared test was used to assess significant association between categories of STYK1 expression and clinicopathological characteristics. For comparing the means of STYK1 expressions among normal, non-invasive and invasive tumors, one way ANNOVA was used. Two tailed P value of less than 0.05 considered significant.

Survival analysis

Follow up data was retrieved for patients. Occurrence of recurrence was defined as locoregional recurrence; at the site of the primary tumor and /or regional LNs while distant metastasis was defined as reappearance of the tumor after start of treatment at any distant site. For survival analysis, Kaplan-Meier analysis and the log-rank test were used for survival analysis. For multivariate analysis, a Cox proportional hazards model was conducted using the patient age, tumor size, tumor grade, and STYK1 expression.

Results

Clinicopathological features of specimens

Age of patients ranged from 28 to 89 years (mean 51 ± 0.8). Tumor sizes ranged from 0.20 cm to 13 cm (mean 3.5 ± 0.2). ER status was positive in 173 (78%) while PR status was positive in 143 (66%). Triple negative phenotypes were detected in 25 (13%) specimens. More details are mentioned in Table 1. When the total number of cases does not reach the total of 220 this is because of the presence of cases with missing data.

STYK1 expression and association with clinicopathological features

STYK1 expression was detected in the cytoplasm of the mammary epithelial cells in normal, In situ and in invasive tumor tissues. No positivity was detected in other tumor components such as the vascular endothelium, stromal cells or in the inflammatory cells. Negative staining was detected in 3 specimens, low expression in 111 (64%) and high expression in 58 (44%) (Fig. 1). STYK1 expression was significantly higher in invasive tumor tissues (Mean 112 ± 4.5) compared to in situ tumor (Mean 55.6 ± 3.3) and to normal lobules (Mean 23.9 ± 1.5), P = 0.001. The same was seen in relation to tumor stage; higher expression was seen in stage III tumors (120 ± 1.57) compared with stage II (mean 106 ± 6) and stage I (mean 99 ± 11.9) tumors, however this association did not reach significant levels P = 0.071 (Fig. 1).

Fig. 1.

Association between STYK1 expression and clinicopathological features in breast cancer. Upper panel: three tissue sections of breast cancer stained immunohistochemically by STYK1 Ab, (A) showing grade 1 (weak) staining intensity (B) showing grade 2 (moderate) staining and (C) showing grade 3 (strong) staining. Lower panel: on the left is a histogram for the frequency of STYK1 expression. In the middle is a mean plot comparing STYK1 means of expression among normal, non-invasive and invasive tumors. On the right, a mean plot comparing STYK1 means of expression among different clinical stages of tumors.

When correlated with clinicopathological features, High expression of STYK1 was significantly associated with tumors with positive vascular invasion P < 0.001, positive LN metastasis P = 0.032, with estrogen receptor positivity, P = 0.033 and with non-triple negative phenotype P = 0.018. No Association was detected with patient age (P = 0.248), tumor size (P = 0.619), tumor grade (P = 0.783), PR reactivity (P = 0.196) or with HER-2 status (P = 0.660). The studied specimens were recategorizes into two categories according to combined high expression of HER‒2 and STYK1. High expression of both STYK1 and HER‒2 score 3 was detected in 16 specimens (10%) while 156 (90%) specimens had low STYK1, OR low HER‒2 OR low both. No significant statistical association was detected between combined high expression and any of the clinicopathological characteristics. More details of association between STYK1 expression and clinicopathological features are summarized in Table 2.

Table 2.

Association between STYK1 expression with clinicopathological characteristics.

	STYK 1 Low expression	STYK1 High expression	P Value		STYK1 Low expression	STYK1 High expression	P Value
Age				Estrogen receptor
≤ 50 Year	51 (44%)	34 (56%)		Negative	31 (26%)	7 (12%)
> 50 Year	65 (55%)	27 (44%)	0.272	Positive	86 (72%)	50 (88%)	0.033
Size				Progesterone receptor
≤ 2 cm	31 (27%)	17 (30%)		Negative	45 (76%)	15 (26%)
> 2 cm	85 (73%)	39 (70%)	0.783	Positive	70 (60%)	42 (74%)	0.196
Stage				HER2 status
I	19 (16%)	4 (7%)		Negative	85 (71%)	40 (66%)
II	57 (49%)	25 (43%)		Positive	27 (23%)	15(24%)	0.660
III	40 (34%)	29 (50%)	0.071
Histopathological tumor types				Tumor grade
NST	93 (81)	53 (82)		I	18 (16%)	8 (14%)
Lobular	12 (11)	9 (15)		II	58 (51%)	33 (58%)
Medullary	3 (2.7)	0		III	36 (32%)	23 (28%)	0.783
Tubulolobular	1 (0.9)	0
Mucinous	2 (2)	0
Papillary	1 (0.9)	0		Vascular invasion
Metaplastic	1 (0.9	1 (1.6)		Negative	79 (70%)	23 (40%)
Micropapillary	1 (0.9)	0		Positive	35 (30%)	35 (60%)	< 0.001
Mixed	3 (2.7)	0	0.447
LN status				Triple negative (TN) phenotype
Negative	37 (32%)	10(17%)		Non TN	86 (83%)	40 (96%)
< 4 LNs	43 (37%)	18 (31%)		TN	18 (17%)	2 (4%)
> 4 LNs	28 (24%)	26 (44%)	0.032				0.018

Note: HER2 = human epidermal growth factor receptor-2, TN=triple-negative breast cancer, LN status= lymph node status. [When the total number of cases does not reach the total of 180 this is because of the presence of cases with missing data].

Survival analysis revealed that 3 ∕ 96 (3%) of the STYK1‒low specimens developed recurrence compared with 7 ∕ 46 (15%) of the STYK1‒high specimens, P=0.011 (Fig. 2). For distant metastasis, 33∕92 (35%) patients of the of the STYK1‒low specimens developed distant metastasis compared with 16∕46 (35%) of the STYK1‒high specimens P=0.830. Time‒dependent ROC curve analysis was done to identify if STYK1 expression is a strong predictor for disease free survival. The analysis revealed that area under the curve was 0.516 with sensitivity and specificity of 0.800 and 0.520, respectively. These findings indicate that STYK1 expression is not a standalone predictor for the disease-free survival.

Fig. 2.

Association between expression of STYK1 with breast cancer outcome. Survival analysis showing significant association between high STYK1 expression with occurrence of local recurrence (A) but not with development of distant metastasis (B) or with overall survival (OS) (C) as seen in the Kaplan Meier survival curves.

Discussion

In the present study, expression of STYK1 was assessed in primary human breast carcinoma tissues. The level of STYK1 expression was found significantly higher in invasive tumors compared with non-invasive tumors indicating role for STYK1 in progression of breast cancer. STYK1 expression was found in similar studies to be higher in cancerous tissue compared with non-cancerous tissue and was recommended to be used as a differentiating marker between normal and malignant tumors in some tumor types^14,15. Our findings along with others’ findings indicate a possible role for STYK1 in early induction of malignant transformation. This is supported by the results reported by Yang and colleagues where enforced expression of STYK1/NOK in transgenic mice significantly predisposed stem cells towards the development of malignant cells¹⁶. The critical step in malignant transformation from non‒invasive to invasive tumors happens when colonies of tumor cell population gain the molecular capabilities to invade through the basement membrane and through the tumoral stromal components. The role of STYK1 in induction of cell invasiveness has been studied in different types of malignant cell lines. Its significant role in induction of invasiveness and migration of malignant tumor cell types was reported in urinary bladder carcinoma⁵, lung carcinoma and renal carcinoma¹⁵. These results were further supported by experimental studies where blocking of STYK1 was found to reduce tumor growth¹⁷. Taken together, these findings suggest that STYK1 has a role in early transformation of mammary epithelial cells from normal to in situ non‒invasive to invasive phenotypes.

The present study did not find association between high STYK1 expression and tumor size. Data from previous research studies identified an association between high STYK1 expression with increased proliferation of cells and therefore with increased tumor size. In a study on different types of malignant cell lines, the protein level of STYK/NOK was found to fluctuate during the cell cycle, with a peak at mitosis and a quick reduction upon mitotic exit¹⁸. This association may be explained by the kinase nature of STYK1. It is well appreciated that proteins with kinase function induce tumor cell proliferation and growth.

Most of the studies which reported association between high STYK1 expression with increased tumor size were invitro studies using cell lines in culture. It is known that testing one molecule at a time in cell culture does not reflect the real microenvironment of tumor biology. Countless other molecular factors contribute to the final functional effect of any molecule. Our findings may indicate a strong role for STYK1 in inducing the invasive capabilities of tumors rather than proliferation and differentiation.

It is known that proteins which have kinase activity are involved in regulation of several biological processes such as proliferation, growth, cell cycle, apoptosis, motility, differentiation and others¹⁹. The most famous examples in breast cancer are the EGFR and ERBB2 (HER‒2). In a recent interesting study, it was found that STYK1 directly interacted with HER‒2 forming a heterodimer complex which results in an increase in the phosphorylation of HER‒2 via the juxtamembrane domain of STYK1. This interaction promote proliferation and migration of breast cancer cells¹¹. In the same study, co-expression of STYK1 and HER‒2 was found correlated with tumor malignant stages. More studies examining HER‒2 and STYK1 reported a crosstalk between STYK1 and HER‒2 in autophagy regulation of HER‒2 and its effects in non-small cell lung cancer^20,21. In the present research study, we were interested to find if there is association between expression of HER2 and STYK1 expressions in our tumor sections. No significant association was seen either between high STYK1 and score 3 + HER2 status or with any HER2 positivity (Scores 1 + and 2+). Similar studies reported the same findings at the mRNA level where no correlation was found between STYK1 and HER‒2 ⁹. It may not be necessary that both molecules are upregulated at the expression detectable level to be identified as positive, instead, they may are functionally acting in a synergetic way.

In the present study, STYK1 was found associated with vascular invasion and with LN metastasis. No data is available in literature about association between STYK1 and vascular invasion in breast carcinoma. Vascular invasion is a complex process in which cross talk among several tumor components, cells and molecules takes place. Experimental studies found that enhanced expression of STYK1 potentiates the migration, invasion, and enhance epithelial mesenchymal transition (EMT) in squamous carcinoma cell lines, thereby promoting metastasis. This was further confirmed by the finding that when STYK1 is, knockdown, inhibition of metastasis is observed²². The same findings were reported in with prostate cancer cell growth²³.

In a study conducted to explain this effect using hepatocellular carcinoma samples, STYK1 was found to stimulate EMT through activation of downstream signaling pathways such as PI3k∕AKT and MAPK∕ERK²⁴. STYK1 overexpression was found to be associated with increased level of other oncogenes and molecules enhance EMT such as Akt (Thr308), p-GSK-3beta (Ser9) and N-cadherin²⁵ which are known to increase invasiveness capabilities of tumor cells. It was found that STYK1 has a role in inducing changes in the tumor vessels in the form of enhancing lymphangiogenesis and causes remodeling of lymphatic vessels during tumor progression²⁶.

A particularly intriguing aspect of our findings is the significant association between STYK1 high expression and estrogen positive tumors compared to estrogen negative ones. Also, the association between this molecule and non‒triple negative tumors was interesting. Many of the markers of aggressive breast cancer behavior are typically enriched in triple negative breast cancer, STYK1 appears to be preferentially upregulated in ER‒positive tumors. This contrasts with the conventional paradigm where ER positivity is often associated with a more favorable prognosis. There is emerging evidence in the last few years suggests that a subset of ER‒positive tumors can exhibit aggressive behavior. The paradoxical pattern of association between STYK1 and ER positivity on one side but the association between high STYK1 expression with poorer survival is not uncommon in breast cancer and other cancers and is not unique for STYK1. Similar associations have been observed for other ER‒related biomarkers such as GATA3 which is typically linked to ER positive status but may confer poorer prognosis when highly expressed. Such phenomenon may be explained by the association of such markers with the more aggressive luminal B breast carcinomas or may be linked to resistance to endocrine therapy. The data in our study is not enough to subcategorize specimens to luminal type A and luminal type B luminal carcinomas. Moreover, the data about patient response to therapy is not identified to find explanation to such paradoxical association related to STYK1 expression. We are currently conducting further data identification and analysis for our cohort to find explanation for such relation.

The prognostic significance for STYK1 was assessed in previous studies where the high expression of STYK1 was associated with poor prognosis in breast cancer and in other types such as lung carcinoma¹³ and gall bladder carcinomas⁶. contradictory findings however were reported on gastric carcinoma where downregulated STYK1 expression correlated significantly with poor tumor differentiation, advanced clinical stage, and poor OS in GC. When STYK1 is expressed at low level, tumors as less likely to have lymph node metastasis²⁵. In our study, although high expression of STYK1 was associated with shorter disease free survival, however by ROC analysis, this expression is of weak predictive value for development of recurrence and therefore cannot be used as a standalone prognostic marker.

This study has some limitations. First it is retrospective single-centre study which may limit the generalizability of the findings to broader breast cancer populations. Second STYK1 expression was assessed using immunohistochemistry which does not allow establishment of a causal relationship. In addition functional essays were not performed to clarify the biological mechanisms underlying the role of STYK1 in tumour invasion and progression. Future studies should include prospective multicentre cohorts to validate the clinical significance of STYK1 expression and improve external validity. Future studies including in vitro and in vivo models is necessary to elucidate the molecular pathways through which STYK1 contributes to vascular invasion and tumour progression. Integration of gene expression, protein expression and pathway analysis may further clarify its oncogenic role.

In conclusion, this study found higher expression of STYK1 in invasive tumors compared with normal and with non-invasive breast carcinoma. This high expression was significantly associated with presence of vascular invasion, LN metastasis and occurrence of recurrence. These findings suggest that STYK1 can be a possible molecular target for breast carcinoma. Targeted therapy which can inhibit STYK1 kinase may be a possible approach for the development of novel therapies in breast carcinoma.

Abbreviations

ER

Estrogen receptor

PR

Progesterone Receptor

EGF

Epidermal growth factor

PDGF/FGF

Proteins and platelet-derived growth factor/fibroblast growth factor

TN breast cancer

triple negative breast cancer

IHC

Immunohistochemistry

LN status

Lymph node status

Author contributions

RM: conception and design, analysis and interpretation of data, lab work and markers analysis, wrote the manuscript.AI: analysis and interpretation of data and substantively revised the manuscript.SA: acquisition of histopathological data and substantively revised the manuscript.AM: acquisition of histopathological data and substantively revised the manuscript.Saj.S. acquisition of clinical, oncological and follow data of patients.Sam.S. lab work and revision of the manuscript.All authors: approved the submitted version and agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

Data availability

The datasets generated and analysed during the current study are available in the [ repository, [figshare] with the following accession link [https://figshare.com/articles/dataset/STYK-1_in_Breast_Carcinoma/31185685?file=61462804]. DOI; [https://doi.org/10.6084/m9.figshare.31185685].

Declarations

Competing interests

The authors declare no competing interests.