Zinc finger protein 639 expression is a novel prognostic determinant in breast cancer

doi:10.21203/rs.3.rs-4130837/v1

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Zinc finger protein 639 expression is a novel prognostic determinant in breast cancer

https://doi.org/10.21203/rs.3.rs-4130837/v1

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Background: ZNF639 is often contained within the overlapping amplicon of PIK3CA, and previous studies suggest that it was involved in the pathogenesis of esophageal and oral squamous cell carcinomas. However, its expression and significance in breast cancer remain uncharacterized.

Methods: Immunohistochemical analysis of ZNF639 was performed on our tissue microarrays. Functional studies including colony formation, transwell cell migration, and in vivo metastasis were performed in breast tumor cells with ZNF639 knockdown by siRNA transfection.

Results: Reduced ZNF639 immunoreactivity was observed in 82% of breast cancer samples independent of hormone receptor and HER2 status. In multivariate Cox regression analyses, ZNF639 expression was negatively associated with recurrence-free survival (hazard ratio = 0.36) and marginally with overall survival (hazard ratio = 0.42). ZNF639 knockdown increased clonogenicity, cell motility, and murine lung metastasis. Snail1, vimentin, and CCL20 expression were upregulated following ZNF639 knockdown, and ZNF639-mediated changes in cell phenotype were neutralized by CCL20 double-knockdown.

Conclusion: Low ZNF639 expression represents a novel prognostic factor for recurrence-free survival in patients with breast cancer.

ZNF639

CCL20

recurrence-free survival

breast cancer

Recent discoveries of genomic alterations have led to a better understanding of the process of carcinogenesis for breast cancer. PIK3CA, encoding the catalytic subunit of phosphoinositide 3-kinases (PI3Ks), is one of the most commonly mutated genes in breast cancer, particularly among luminal subtypes [1, 2]. The dysregulation of PI3Ks modulates the activation of the serine/threonine kinase AKT and a range of downstream effectors that promote tumor initiation and progression. PI3K inhibitors such as alpelisib have shown clinical benefits in progression-free survival among patients with hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative, PIK3CA-mutated, advanced or metastatic breast cancer [3, 4].

Oncogenic activation of the PI3K/AKT pathway largely results from activating mutations of PIK3CA; nonetheless, other mechanisms, including PIK3CA amplification and loss of function of the pathway down-regulator phosphatase and tensin homolog (PTEN), have been reported. A study on the genomic landscape of circulating tumor DNA in patients with metastatic breast cancer revealed that the most frequent copy number variants were MYC, FGFR1, and PIK3CA [5]. Additional genes are located within the overlapping 3q26 amplification region of PIK3CA. Among these, ZNF639, also known as ZASC1 (zinc finger amplified in esophageal squamous cell carcinoma 1), is often contained within the same amplicon of PIK3CA [6]. ZNF639 has previously been associated with the pathogenesis of esophageal and oral squamous cell carcinomas [7, 8]. However, its oncogenic role in breast cancer has not been investigated.

In the present study, we aimed to explore the clinical and biological significance of ZNF639 expression in breast cancer. We set out to examine its expression and clinicopathological implications using immunohistological analyses as well as cellular and murine experiments.

Patients

The construction of tissue microarrays has been described in detail previously [9]. In brief, female patients who underwent mastectomy for breast cancer between 2004 and 2010 were randomly selected from the pathology archives of MacKay Memorial Hospital. The diagnostic H&E slides were reviewed, and representative areas were chosen to annotate three tissue spots per case. Lost cores were excluded from analysis. Finally, a total of 222 patients (tumor-node-metastasis [TNM] stage I, n = 29; stage II, n = 114; stage III, n = 73; stage IV, n = 6) were included in the present study. This study was approved by the Institutional Review Board (11MMHIS154).

Immunohistochemical analysis

Five-µm sections were taken from the recipient tissue microarray blocks and subjected to deparaffinization, rehydration, and antigen retrieval. Immunohistochemical staining was performed by incubation with sections with rabbit polyclonal anti-ZNF639 antibody (NBP2-15177; Novus Biologicals, Centennial, CO, USA) diluted to 1:100 at 4° C overnight. DAB (Dako) was used for color development and hematoxylin for counterstaining.

ZNF639 expression was quantified according to the intensity and extent of immunoreactivity by two independent researchers as described in our protocol [10]. The scores ranged from 0 (no staining), 1+ (weak staining), 2+ (moderate staining) to 3+ (strong staining). Scores from three cores of the same patient were averaged. An average score higher than or equal to 2 was considered to be high ZNF639 expression.

Cell culture and transfections

Human breast tumor cell lines MCF-7 and MCF-10A were purchased from the American Type Culture Collection, Manassas, VA, USA [11]. MCF-7 cells were cultured in Eagle's minimum essential medium supplemented with 2 mM glutamine, 1% non-essential amino acids, and 10% fetal bovine serum. MCF-10A cells were maintained in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 supplemented with 10 µg/mL insulin, 20 ng/mL epidermal growth factor, 500 ng/mL hydrocortisone, 100 ng/mL cholera toxin, and 5% horse serum.

ZNF639 siRNAs were obtained from Sigma-Aldrich, St. Louis, MO, USA. Transfection of ZNF639 siRNAs was performed using the Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Effects of siRNA transfections were tested after incubation for 48 hours.

Colony-formation assay

For the colony-forming assay, 700 MCF-7 cells or 300 MCF-10A cells were seeded into six-well plates in complete culture medium. After 7 days, colonies were stained with 3% crystal violet and examined under microscopy [12]. Colonies that contained at least 50 individual cells were counted.

Transwell migration assay

Breast tumor cells (MCF-7, 6 × 10⁴ cells/insert; MCF-10A, 1 × 10⁴ cells/insert) were plated into transwell inserts of polycarbonate filters with 8-µm pores (Corning Life Sciences, Corning, NY, USA) [13]. Cells in the transwell insert were allowed to migrate towards the underside of the membrane for 48 hours. Cells on the lower surface of the filter were stained with Diff-Quik (Sysmex, Kobe, Japan) and counted from five random fields under microscopy.

Western blotting

Proteins extracted from cell lysates were subjected to electrophoresis on polyacrylamide gels, followed by transfer to a nitrocellulose membrane. The membrane was blocked in 5% skim milk for 1 hour and incubated with the following primary antibodies [14]: anti-ZNF639 (ab185106; Abcam, Cambridge, UK), anti-Snail1 (PAB1924; Abnova, Taipei, Taiwan), anti-vimentin (V6630; Sigma-Aldrich), anti-CCL20 (GTX31213; GeneTex, Irvine, CA, USA), anti-phospho-AKT Ser473 (#9271; Cell Signaling Technology, Danvers, MA, USA), anti-AKT (#4691; Cell Signaling Technology), anti-phospho-STAT3 Tyr705 (#9131; Cell Signaling Technology), anti-STAT3 (#9132; Cell Signaling Technology), and anti-β-actin (A5441; Sigma-Aldrich). The protein bands were detected with the SuperSignal West Pico enhanced chemiluminescent reagent (Thermo Fisher Scientific).

Oncology proteome profiler

Cell lysates were prepared from MCF-10A cells transfected with a control siRNA or ZNF639 siRNA. The expression of cancer-related proteins was evaluated using the Proteome Profiler Human XL Oncology Array (ARY026; R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

KM-plotter analysis

The KM-plotter is an online platform integrating available microarray and RNA-seq data with clinical characteristics of individual patients [15]. Data from gene chip studies and RNA-seq studies were separately analyzed with the option set to 'auto select best cutoff' and 'no subtype restriction.'

In vivo metastasis assay

Animal studies were approved by the institutional animal care and use committee of MacKay Memorial Hospital. Six-week-old NOD/SCID mice were purchased from the National Laboratory Animal Center, Taipei, Taiwan. The mice received subcutaneously implanted pellets containing 0.72 mg 17β-estradiol 3 days prior to injection of MCF-7 cells transfected with a control siRNA or ZNF639 siRNA (n = 5, each group) via the tail vein. Seven weeks after tail vein injection, the mice were sacrificed, and the harvested lungs were subjected to H&E examination and GATA3 immunostaining.

Statistical analysis

For survival analysis, Kaplan-Meier curves and the log-rank test were used to assess statistical significance. Cox regression analysis was used to identify the factors independently associated with overall and recurrence-free survival. Experimental data were analyzed using unpaired t-tests or one-way ANOVA with post-hoc Tukey's multiple comparison testing. Data were presented as the mean ± standard deviation, and P < 0.05 denoted significant differences between the groups.

Downregulation of ZNF639 associated with unfavorable outcomes

To explore the clinical significance of ZNF639 expression in breast cancer, we queried the KM-plotter platform for breast cancer patients who had available ZNF639 expression data. Of note, both integrated microarray and RNA-seq studies showed that patients with low ZNF639 expression had worse overall survival compared to those with high ZNF639 expression (Fig. 1a). Next, we performed immunohistochemical analysis of our tissue microarrays. Cytoplasmic and nuclear ZNF639 expression was observed in glandular epithelial cells of normal and tumorous breast tissues. Nonetheless, ZNF639 expression was attenuated in the majority of breast cancer samples (Fig. 1b). When we dichotomized the patient cohort into low- and high-expression groups using a cutoff score of 2, 39 (18%) of the 222 patients retained high ZNF639 expression in the tumor area. There was no difference in TNM stage (P = 0.724), estrogen receptor positivity (P = 0.591), progesterone receptor positivity (P = 0.218), or HER2 status (P = 0.349) between the two groups. Consistent with the KM-plotter data, patients with low ZNF639 expression had significantly shorter overall (P = 0.024) and recurrence-free survival (P = 0.014) during follow-up (Fig. 1c).

To determine whether ZNF639 expression was an independent prognostic factor in breast cancer, multivariate Cox regression analyses were performed. As shown in Table 1, TNM stage (hazard ratio [HR] = 3.22) and age (HR = 1.03) were independent predictors for overall survival. ZNF639 score was negatively associated with overall survival (HR = 0.42) but did not reach statistical significance. TNM stage (HR = 2.97) remained the major determinant of recurrence-free survival. Importantly, the ZNF639 score (HR = 0.36) was significantly associated with worse recurrence-free survival in our patients with breast cancer.

Table 1

Multivariate Cox regression analysis of overall and recurrence-free survival in 222 patients with breast cancer
	Overall survival		Recurrence-free survival
	Hazard ratio (95% CI)	P-value	Hazard ratio (95% CI)	P-value
Age	1.03 (1.00-1.05)	0.017	0.99 (0.97–1.01)	0.459
Stage	3.22 (2.20–4.71)	< 0.001	2.97 (1.93–4.58)	< 0.001
Grade	1.23 (0.76–2.01)	0.397	1.07 (0.66–1.74)	0.793
ER	1.12 (0.57–2.17)	0.744	0.91 (0.45–1.83)	0.788
PR	0.60 (0.31–1.16)	0.127	0.75 (0.38–1.49)	0.417
HER2	0.72 (0.42–1.24)	0.234	0.72 (0.42–1.26)	0.254
ZNF639	0.42 (0.16–1.05)	0.064	0.36 (0.14–0.90)	0.028

Clonogenicity and cell migration potentiated by ZNF639 knockdown

We used four different siRNA constructs to reduce ZNF639 expression in breast tumor cell lines MCF-7 and MCF-10A (Fig. 2a). Interestingly, ZNF639 siRNA #2 and #4 consistently increased colony formation in both cell lines (Fig. 2b). In the meantime, transfection with siRNA #4 significantly boosted transwell cell migration by 69% and 45% in MCF-7 and MCF-10 cells, respectively (Fig. 2c). These results suggest that ZNF639 may play a role in the regulation of clonogenic survival and cell motility in breast tumor cells.

ZNF639 knockdown promoting metastasis in vivo

We chose siRNA #4 to examine the in vivo effect of ZNF639 knockdown in NOD/SCID mice. In agreement with our in vitro observations, tail vein injection of MCF-7 cells transfected with ZNF639-targeting siRNA resulted in a significantly higher burden of lung metastasis compared with the control group (Fig. 2d). Furthermore, we observed that ZNF639 knockdown increased Snail1 expression, an epithelial-to-mesenchymal transition regulator, in both cell lines (Fig. 3a). The expression of vimentin, the major subunit of the intermediate filaments involved in cell movement, was also upregulated following ZNF639 silencing. These results suggest that ZNF639 knockdown renders breast tumor cells more motile and facilitates in vivo metastasis.

CCL20 upregulation in response to ZNF639 knockdown

The oncology proteome profiler was used to identify potential regulators involving ZNF639-mediated modulation of cell motility. Notably, C-C chemokine ligand 20 (CCL20), or macrophage inflammatory protein-3α (MIP-3α), showed the greatest increase in protein expression following ZNF639 knockdown (Fig. 3b). The upregulation of CCL20 was further validated using immunoblotting. Given that CCL20 is the ligand for the C-C chemokine receptor CCR6, we examined downstream targets along the CCR6 signaling pathway. Concordantly, we observed an increase in phosphorylation of AKT and STAT3 following siRNA knockdown of ZNF639 (Fig. 3c). Taken together, CCL20 upregulation ensues after ZNF639 knockdown in breast tumor cells.

ZNF639 silencing neutralized by CCL20 double-knockdown

To determine the dependency of CCL20 upregulation on ZNF639-mediated changes in cell phenotype, breast tumor cells with ZNF639 knockdown were subsequently transfected with a second siRNA targeting CCL20. As shown in Fig. 4a, the second knockdown reduced the increased expression of CCL20 induced by ZNF639 knockdown. Additionally, ZNF639 knockdown-induced increases in clonogenicity and cell motility were effectively abolished by CCL20 silencing (Fig. 4b and 4c). Collectively, these findings suggest that the tumor-promoting effects of ZNF639 knockdown may be partly mediated by CCL20 upregulation.

Zinc finger proteins are the largest transcription factor family in the human genome [16]. Zinc finger proteins can act as an oncogene or tumor suppressor dependent on cellular context. A recent study surveyed the prognostic value of 63 cancer-associated ZNF family genes in breast cancer using the Cancer Genome Atlas database [17]. However, ZNF639 was not included in the analysis. Bogaerts and colleagues identified ZNF639 orthologs in humans, Xenopus, mice, rats, and chickens, all of which showed highly conserved sequences, especially in the C-terminal region [18]. Nuclear expression of the endogenous ZNF639 protein was demonstrated in MCF-7/AZ cells using immunocytochemical analysis, and luciferase reporter assays revealed that ZNF639 is a transcriptional repressor [18]. ZNF639 appears to participate in regulating the anchoring of E-cadherin to the cytoskeleton through α-2-catenin.

Japanese investigators first disclosed that this Kruppel-like transcription factor was overexpressed in esophageal squamous cell carcinoma [7]. Exogenous expression of ZNF639 promoted the growth of esophageal cancer cells, while ZNF639 downregulation suppressed growth [7]. Subsequently, increased mRNA expression of ZNF639 was reported in oral squamous cell carcinoma [8]. Although ZNF639 mRNA expression was not correlated with clinical stages in oral cancer, high ZNF639 mRNA expression was associated with lymph node metastasis. By contrast, in this study, we demonstrated that low ZNF639 expression was significantly associated with disease recurrence in breast cancer. Integrated public databases further suggest that low ZNF639 expression was linked to lower overall survival among breast cancer patients compared to those with high ZNF639 expression. This discrepancy indicates that ZNF639 may function in a context-dependent, cancer-type-specific manner. In a study comparing BRAF inhibitor-sensitive and -resistant melanoma cells, ZNF639 mRNA expression was consistently downregulated in both the resistant 2D and 3D cultures [19]. This finding implies that ZNF639 may also be involved in treatment resistance in some circumstances.

The mechanisms underlying tumor biology regulated by ZNF639 are poorly understood. Results from the ZNF639-knockout model suggest that ZNF639 is important for myeloid cell differentiation in the bone marrow compartment [20]. Furthermore, ZNF639 was shown to bind to specific DNA binding sites in the murine leukemia virus promoter and was required for efficient viral mRNA transcription [21]. In the current study, we found that ZNF639 knockdown increased the expression of CCL20 and downstream phosphorylation of AKT and STAT3. CCL20 is one of the C-C and C-X-C chemokines that correlate with the clinical outcomes of breast cancer patients [22]. Our findings are in agreement with others showing that CCL20 induces epithelial-mesenchymal transition with upregulation of vimentin and Snail1 in breast epithelial cells through the PI3K/AKT and JAK2/STAT3 pathways [23, 24]. In addition, CCL20 has been reported to mediate taxane resistance in breast cancer stem cells [25]. In this regard, the link between ZNF639 downregulation and CCL20 upregulation deserves further critical evaluation.

We acknowledge several limitations of this study. Genetic information of our patients was not available. Therefore, the prevalence of PIK3CA mutation and its association with ZNF639 expression are unknown. Second, our cell model was limited to MCF-7 and MCF-10A cells. Whether ZNF639 exhibits similar functions in other subtypes of breast cancer cells, e.g., the triple-negative subtype, has not been determined. Finally, the molecular relationships between ZNF639 and CCL20 remain elusive. Despite these limitations, our study for the first time investigated the expression and clinical prognostication of ZNF639 in breast cancer. Contrary to previous reports, ZNF639 may act as a tumor suppressor, and its downregulation has been associated with compromised outcomes in patients with breast cancer irrespective of hormone receptor and HER2 status.

In conclusion, our research shows that expression of ZNF639 is reduced in some patients with breast cancer and that low ZNF639 expression is an independent prognostic factor for recurrence-free survival. Knockdown of ZNF639 in breast tumor cells promotes clonogenicity and cell migration as well as in vivo metastasis. These effects are at least partially mediated by the upregulation of CCL20. Further studies are needed to evaluate its role in treatment resistance and underlying mechanisms related to breast cancer pathogenesis.

Acknowledgments

We are grateful to Pou-Chu Pun, MS, for her assistance in constructing tissue microarrays.

Author contributions

Conceptualization: Fang Lee, Yuan-Ching Chang

Methodology: Yi-Min Liu, Shao-Chiang Chang

Formal analysis and investigation: Ming-Jen Chen, Wen‐Chien Huang

Writing - original draft preparation: Fang Lee, Shih-Ping Cheng

Writing - review and editing: Yuan-Ching Chang, Shih-Ping Cheng

Funding

This work was supported by research grants from MacKay Memorial Hospital (MMH-E-112-08 and MMH-E-113-08).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflict of interest

The authors declare no conflict of interest.

Research involving human participants and/or animals

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. For this type of study, formal consent is not required. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

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Version 1

posted

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Zinc finger protein 639 expression is a novel prognostic determinant in breast cancer

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Patients

Immunohistochemical analysis

Cell culture and transfections

Colony-formation assay

Transwell migration assay

Western blotting

Oncology proteome profiler

KM-plotter analysis

In vivo metastasis assay

Statistical analysis

Results

Downregulation of ZNF639 associated with unfavorable outcomes

Clonogenicity and cell migration potentiated by ZNF639 knockdown

ZNF639 knockdown promoting metastasis in vivo

CCL20 upregulation in response to ZNF639 knockdown

ZNF639 silencing neutralized by CCL20 double-knockdown

Discussion

Declarations

Acknowledgments

Author contributions

Funding

Data availability

References

Status:

Version 1