Correlation and clinical significance of hepcidin and STAT3 in colorectal cancer

Abstract

BACKGROUND

Iron homeostasis, essential for many life processes, is maintained through a regulatory network centered on liver-synthesized hepcidin. Hepcidin controls iron output by binding to transferrin while performing multiple functions, including immune regulation. Colorectal cancer (CRC), a highly prevalent malignant tumor, is closely associated with iron metabolism disorders.

AIM

To investigate the expression profiling and characteristics of hepcidin and the signal transducer and activator of transcription 3 (STAT3), iron metabolism indicators, in patients with CRC.

METHODS

The surgically resected pathological specimens of 58 CRC patients in the Guangzhou First People’s Hospital and the Second Affiliated Hospital of Guangzhou Medical University (January 2023 to January 2025) were collected for analysis. The adjacent normal intestinal mucosa tissues of the 20 patients with CRC served as the control group. Hepcidin and STAT3 underwent immunohistochemistry for expression quantification. The correlation between hepcidin, STAT3, and clinicopathological features was analyzed.

RESULTS

The hepcidin and STAT3 positive expression rates were statistically elevated in CRC tissues compared to normal intestinal mucosa tissues (67.2% and 63.8% vs 20.0% and 25.5%, P < 0.01). Hepcidin expression correlated markedly with T stage (P < 0.01), with statistical significance identified between well- and poorly differentiated groups (P < 0.01); it was slightly related to lymph node metastases, distant metastases, age, or gender. STAT3 expression varied statistically among patients with different lymph node metastasis (LNM) status and T staging (P < 0.01), with significant differences noted between poorly and well differentiated groups (P < 0.01); it showed no marked link with distant metastasis, age, and sex. Hepcidin and STAT3 were positively correlated (P < 0.01).

CONCLUSION

Hepcidin and STAT3, showing significantly upregulated expression in CRC tissues, were related to T staging. The two correlate with CRC occurrence and development, showing potential for predicting tumor malignancy extent, staging, and LNM status.

Key Words: Hepcidin; STAT3; Colorectal cancer; Clinical significance; Lymph node metastasis

Core Tip: Iron metabolism disorders play a pivotal role in colorectal cancer (CRC) development and progression, with this disordered state closely associated with hepcidin and signal transducer and activator of transcription 3 (STAT3) dysregulation. As a vital transcription factor, STAT3, upon activation, translocates to the cell nucleus and directly binds to the hepcidin gene promoter region, thereby upregulating hepcidin expression. Concurrently, CRC cells adapt to iron overload environments by modulating the expression of iron metabolism-related proteins. This study aims to investigate the expression profiling and characteristics of hepcidin and STAT3, iron metabolism indicators, in patients with CRC.

INTRODUCTION

Globally, colorectal cancer (CRC) ranks third in incidence among cancers and second in cancer-associated deaths. With a steadily rising incidence in China, CRC ranks second among newly diagnosed cancer cases in the country. Studies have strongly linked tumors to systemic iron homeostasis disorders. Hepcidin is a key regulator of iron metabolism. The hepcidin promoter site -64/-72 is the signal transducer and activator of transcription 3 (STAT3)-binding motif. Interleukin (IL)-6 regulates hepcidin synthesis through the Janus kinase (JAK)-STAT3 axis. This study employed immunohistochemistry (IHC) to quantify hepcidin and STAT3 expression in the pathological tissue specimens of 58 patients with CRC. How hepcidin, STAT3, and clinical characteristics correlated was also investigated.

MATERIALS AND METHODS

Ethics statement

This study was approved by the Ethic Committee of the Guangzhou First People’s Hospital (Approval No. K-2023-145-02). All participants, including CRC cases and healthy volunteers, provided written informed consent.

Human samples

This study selected 58 patients, 37 males plus 21 females with a mean age of 63 years, who received surgical resection for CRC in the Guangzhou First People’s Hospital and the Second Affiliated Hospital of Guangzhou Medical University (January 2023 to January 2025). The diagnosis was pathologically confirmed, and no chemotherapy or radiotherapy was performed before the operation. By referring to the National Colorectal Cancer Cooperative Group TNM staging standard, stages T1-T2 and T3-T4 CRC were confirmed in 21 and 37 cases, respectively. While 25 cases had lymph node metastasis (LNM), 13 developed distant metastasis. The control group comprised adjacent normal intestinal mucosa tissues collected from 20 CRC patients. Adjacent normal intestinal mucosa samples serving as controls were collected from regions located at least 5 cm from the macroscopic tumor margin. This distance criterion was established based on clinical practice guidelines and the existing literature to minimize the risk of microscopic tumor infiltration or molecular alterations in the control tissues. Only tissues unanimously confirmed by both pathologists as histologically normal (showing intact architecture and no tumor cells) were included as controls. Figure 1 shows the study flow chart.

Figure 1 Flow chart of the study. CRC: Colorectal cancer; STAT3: Signal transducer and activator of transcription 3.

IHC

Hepcidin expression was detected by IHC staining (OBiO Technology, Shanghai). All paraffin specimens were sliced into 4 μm sections. Notably, 4 consecutive slices were selected, oven baked at 60 °C for 30 minutes, dewaxed with xylene and different alcohol concentrations, and then hydrated with tap water for 2 minutes. One slide was stained with hematoxylin and eosin for pathological diagnosis, and the other 3 slides were stained using streptavidin-Biotin complex IHC. STAT3 expression was detected by IHC staining (OBiO Technology, Shanghai). All paraffin specimens were sliced into 4 μm sections. Notably, 4 consecutive slices were selected, oven baked at 68 °C for 1 hour, deparaffinized with xylene, hydrated with gradient ethanol, heat-repaired with antigen in a pressure cook, blocked with hydrogen peroxide (3%), and finally incubated overnight with primary antibodies at 4 °C. The slices underwent PBS rinsing for 3 minutes × 3, followed by a room-temperature culture with secondary antibodies for 15 minutes and then re-rinsed with PBS (3 minutes × 3). After streptomyces avidin-peroxidase addition, samples underwent a 15 minutes incubation at room temperature and PBS rinsing (3 minutes × 3). DAB served as a chromogenic reagent. Known slides served as positive controls, while PBS was used in place of the primary antibody as a negative control.

IHC score

Two independent pathologists blinded to clinical data and patient outcomes performed IHC scoring. Discordant cases (> 10% score difference) were re-evaluated jointly to reach consensus. This approach minimized observer bias and enhanced reproducibility. The IHC score was calculated as positive cell count × color intensity under five high-power fields. Positive cells were defined as clear brown-yellow granules appearing in the cytoplasm or nucleus. Details: (1) The positive cell count was scored from 0 to 4: (0: No positive cells; 1: ≤ 25%; 2: 26%-50%; 3: 51%-75%; and 4: > 75%); and (2) The score range for staining intensity was 0-3 (0: Colorless; 1: Light yellow; 2: Brown yellow; and 3: Sepia). By multiplying the two index scores, the IHC score was obtained, which was divided into four grades: 0 was negative (-), 1-4 was weak positive and marked as (+), 5-8 was moderately positive (++), and > 8 was strongly positive (+++).

Statistical analysis

Data analyses were implemented in SPSS17.0 (IBM, Armonk, NY, United States). A χ² test was used to compare the differences of related factors, and Spearman’s rank correlation identified inter-variable correlations. A P < 0.01 served as the significance threshold.

RESULTS

Hepcidin expression in CRC tissues

CRC tumor tissues exhibited higher hepcidin expression than normal intestinal mucosa tissues (P < 0.01). The positive expression rate was 20.0% (4/20) in normal intestinal mucosa tissues vs 67.2% (39/58) in CRC tissues. Hepcidin presented markedly upregulated levels in cases with higher T stages (42.9% in T1-T2 CRC vs 81.1% in T3-T4 disease; P < 0.01). The well- and poorly-differentiated groups also showed statistical differences in hepcidin expression (57.1% vs 88.9%, P < 0.01). However, no notable differences in hepcidin positive expression rates were identified between LNM and non-LNM patients (72.0% vs 63.6%, P > 0.05), or between those with distant metastasis vs patients without (69.2% vs 66.7%, P > 0.05). Hepcidin showed no significant correlation between age and gender (Figure 2 and Table 1).

Figure 2 Immunohistochemistry staining for hepcidin or signal transducer and activator of transcription 3. A: Negative control of hepcidin; B: Positive control of hepcidin; C: Negative control of signal transducer and activator of transcription 3 (STAT3); D: Positive control of STAT3.

Table 1 Expression of hepcidin and signal transducer and activator of transcription 3 in colorectal cancer tissues and their correlation with clinical characteristics.

Characteristic	N	Hepcidin			STAT3
		Positive cases	Positive rate (%)	P value	Positive cases	Positive rate (%)	P value
Group
CRC	58	39	67.2	< 0.001	37	63.8	< 0.001
Control	20	4	20.0		5	25.0
T stage
T1 and T2	21	9	42.9	< 0.001	10	47.6	0.001
T3 and T4	37	30	81.1		27	73.0
Differentiation
Well	7	4	57.1	0.0071	3	42.9	0.0011
Medium	42	27	64.3		25	59.5
Low	9	8	88.9		9	100
N
Yes	25	18	72.0	0.312	19	76.0	0.003
No	33	21	63.6		18	54.5
M
Yes	13	9	69.2	0.186	9	69.2	0.867
No	45	30	66.7		28	62.2
Age (year)
< 65	14	10	71.4	0.271	9	64.3	0.673
≥ 65	44	29	66.0		28	63.6
Gender
Male	37	25	67.6	0.566	24	64.9	0.438
Female	21	14	66.7		13	61.9

¹Comparison between the well-differentiated group and the low-differentiated group.

STAT3: Signal transducer and activator of transcription 3; CRC: Colorectal cancer; N: Lymph node metastasis; M: Distant metastasis.

STAT3 expression in CRC tissues

STAT3 was expressed in the cytoplasm. The positive STAT3 expression rate reached statistical significance between normal intestinal mucosa [25.0% (5/20)] and CRC tissues [63.8% (37/58); P < 0.01]. The significance was still established between stage T1-T2 vs T3-T4 patients (47.6% vs 73.0%; P < 0.01), and between the well and poorly differentiated groups (42.9% vs 100%; P < 0.01). STAT3 expression was obviously increased in patients with LNM relative to those without (76.0% vs 54.5%, P < 0.01). In patients with and without distant metastasis, however, no such difference was observed (69.2% vs 62.2%; P > 0.05). Age and gender did not lead to noticeable variations in STAT3 levels.

Hepcidin-STAT3 correlation

Among the 37 cases with positive hepcidin expression, 33 were STAT3-positive and 4 were STAT3-negative, whereas 4 were STAT3-positive and 15 were negative of the 19 cases with negative hepcidin expression. Spearman’s rank test revealed a positive link between hepcidin and STAT3 expression (P < 0.01; Table 2).

Table 2 Correlation between the expression of hepcidin and signal transducer and activator of transcription 3 in colorectal cancer tissues.

Hepcidin	STAT3			P value
	N	Positive	Negative
Positive cases	37	33	4	< 0.01
Negative cases	19	4	15

STAT3: Signal transducer and activator of transcription 3.

DISCUSSION

CRC is a common malignancy. Iron metabolism strongly correlates with tumor occurrence, development, invasion, and metastasis. Numerous epidemiological studies have linked cancer to systemic iron homeostasis disorders[1], which are mainly reflected in iron metabolism indicators and the expression of genes that regulate iron metabolism. Hepcidin is a key molecular regulator of iron homeostasis. Hepcidin reduces the iron level and maintains the normal iron homeostasis of the body by inhibiting intestinal iron absorption and iron release from macrophages and aging red blood cells[2]. The hepcidin-malignancy relationship has now been gradually revealed in breast[3-6] and non-small cell lung cancers[7]. Zuo et al[8] studied hepcidin protein expression in gastric cancer using IHC and revealed higher expression in gastric cancer tissues than in normal adjacent gastric mucosa tissues and a positive association of hepcidin protein expression with T staging. During inflammation, infection, and cancer, iron is redistributed from the circulation to hepatocytes and macrophages for storage, which limits iron availability to invading microorganisms and tumor cells[9]. Close relationships were found between hepcidin and tumors. Hepcidin, via hepcidin-FPN axis-mediated iron regulation, can promote tumor cell growth and invasiveness.

As a common inflammatory cytokine, IL-6 is overexpressed in tumors and regulates cell proliferation and differentiation through several signal transduction[10-12]. The IL-6-STAT3 signaling pathway regulates hepcidin synthesis. Therefore, increasing attention has focused on the roles of the iron metabolism indicators hepcidin and inflammatory cytokines in tumor development and on their interactions. In this study, we found markedly increased hepcidin expression in CRC tissues. Statistically elevated hepcidin expression was also observed in high T stage (T3 and T4) vs low T stage (T1 and T2), and in poorly vs well-differentiated tumors, linking hepcidin levels to tumor invasion depth and differentiation degree. Hepcidin expression is significantly elevated in CRC, as confirmed by multiple studies. Its expression levels in tumor tissues are markedly higher than in adjacent normal tissues and are closely associated with tumor initiation and progression. A study published in Nature Metabolism indicates that hepcidin is abnormally activated in CRC epithelial cells[13]. By inhibiting ferroportin, a protein responsible for iron efflux, it promotes intracellular iron accumulation. This supports nucleotide synthesis and mitochondrial function, thereby driving tumor cell proliferation. Hepcidin expression is closely associated with tumor differentiation. Despite the lack of direct quantitative studies, the underlying pathological mechanisms suggest higher expression in poorly differentiated tumors. Poorly differentiated CRC cells lose normal glandular architecture, exhibit high metabolic activity and invasiveness, and demand greater reliance on iron-dependent metabolic pathways (e.g., nucleotide synthesis)[13]. Hepcidin supports this high-metabolic phenotype by regulating iron homeostasis, making its upregulation likelier in poorly differentiated tumors. However, hepcidin showed no marked association with distant metastasis, LNM, age, or gender; nevertheless, the small number of patients with distant metastasis in this cohort warrants further study.

STAT3 overexpression or persistent activation in malignancies observed in recent studies suggests a close relationship between STAT3 signaling pathway activation and tumorigenesis[14]. In the inflammatory response, IL-6 and other cytokines induce hepcidin transcription by activating STAT3 signaling to the hepcidin gene promoter. IL-6 binding to its receptor induces the phosphorylation of the intracellular signaling molecule STAT3. The phosphorylated STAT3 forms a dimer and is transferred to the nucleus, where it binds to the corresponding site on the hepcidin promoter to induce hepcidin expression[15]. IL-6 exhibits increased expression in gastric cancer tissues[10-12], while, in colon cancer, high IL-6 levels serve as a predictor for poor survival and prognosis[16]. The IL-6-JAK2-STAT3 axis plays a crucial role in the malignant transformation of normal cells in breast cancer[17]. Our previous study uncovered the IL-6-STAT3 signaling modulation of Th17 cell differentiation in MDS patients with iron overload. To date, STAT3 expression in CRC has rarely been reported. In this study, we found an obviously elevated positive expression rate of STAT3 in CRC tissues. STAT3 is highly expressed in patients with high T stage (T3 and T4) CRC. STAT3 was highly expressed in the poorly differentiated group. Higher positive expression rates of STAT3 were also determined in patients with LNM than in those without. STAT3 was correlated with tumor invasion depth, differentiation degree, and LNM. Hence, STAT3 is involved in CRC progression. STAT3 expression detection may aid in tumor progression assessment and clinical treatment guidance, with potential utility as a promising indicator of CRC onset and development. Our study showed no significant correlations between STAT3 levels and distant metastasis, age, or gender. Notably, the observed positive hepcidin correlation with STAT3 expression indicates that STAT3-hepcidin signal transduction pathway activation may be related to CRC occurrence and progression.

However, our study has several limitations that warrant consideration. First, the sample sizes within certain clinically important subgroups were small. While these subgroup analyses were planned a priori to explore the potential heterogeneity of treatment effects or prognostic factors across these key biological strata, the limited sample sizes inevitably constrain the statistical power. This reduces precision (wide confidence intervals) and increases the type II error risk. The results in these subgroups should be interpreted cautiously. Before verification with a larger sample size, its applicability to a broader population is limited. These analyses serve to inform future validation studies rather than to guide clinical decisions. Furthermore, the molecular mechanisms underlying STAT3-mediated promotion of CRC require further investigation. Although our data support the biologically plausible hypothesis that STAT3 may regulate hepcidin expression through IL-6 signaling, we acknowledge that circulating or tissue-specific IL-6 levels were not directly assessed in the current study. Future investigations evaluating IL-6 expression dynamics alongside STAT3 activation status would support the characterization of this regulatory axis. Such work could mechanistically clarify whether STAT3-driven hepcidin modulation in CRC operates primarily via IL-6-dependent pathways or involves alternative compensatory mechanisms.

This study still has limitations; the small sample size may have substantially reduced the statistical power, thereby increasing the risk of erroneous conclusions. During the preliminary exploration phase, small samples facilitate hypothesis generation and effect size estimation, providing a foundation for subsequent large-sample studies. Therefore, a large-sample controlled experiment is required to further validate the conclusions of this research.

CONCLUSION

Hepcidin and STAT3 levels significantly increased in CRC tissues, which were related to tumor T stage, tumor invasion depth, and differentiation degree. Hepcidin was positively correlated with STAT3. Combined hepcidin + STAT3 detection shows potential as an objective indicator reflecting CRC cell biological behaviors. STAT3-hepcidin signal transduction pathway activation may be associated with CRC occurrence and progression.