KAISO Promotes Poor Prognosis in Hepatocellular Carcinoma Patients by Enhancing Neutrophil Infiltration via IGFBP1

doi:10.21203/rs.3.rs-4820754/v1

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KAISO Promotes Poor Prognosis in Hepatocellular Carcinoma Patients by Enhancing Neutrophil Infiltration via IGFBP1

https://doi.org/10.21203/rs.3.rs-4820754/v1

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Background

KAISO is a transcriptional regulator involved in gene expression, cell proliferation, and apoptosis, linked to cancer prognosis and tumor aggressiveness, making it a potential bi-omarker and therapeutic target.

Methods: We used bioinformatics analyses to evaluate KAISO expression and its effect on survival prognosis across 33 types of pan-cancer. We also examined the link between KAISO expression and immune cell infiltration. To investigate the control of down-stream proteins by KAISO, we used dual-luciferase reporter assays, electrophoretic mobility shift assays (EMSA), and chromatin immunoprecipitation (ChIP). Additionally, we validated the role of KAISO in regulating immune cell infiltration using a subcutaneous tumor model in animals and human tumor samples.

Results: Our research revealed that KAISO is crucial in regulating the growth and progression of various malignancies, including hepatocellular carcinoma (HCC). We demonstrated that high KAISO expression is associated with poor prognosis in HCC. KAISO was found to regulate the transcription of IGFBP1 and neutrophil infiltration and influence HCC pro-liferation through cell cycle-related molecular pathways. Finally, we confirmed that reducing KAISO expression can inhibit neutrophil infiltration and tumor growth.

Conclusion: Our findings suggest that KAISO could be an important biomarker and molecular target for HCC patients.

KAISO

IGFBP1

HCC

Neutrophil

Liver cancer is the third leading cause of death related to cancer and the sixth most common type of cancer worldwide [1, 2]. HCC accounts for more than 80% of liver cancer patients [3, 4]. The development of HCC is marked by complex biological processes, such as the disruption of tumor suppressor genes and oncogenes, abnormal activation of molecular signaling networks, alteration of HCC cell differentiation, and stimulation of angiogenesis [5, 6]. Therefore, it is essential to clarify the precise molec-ular pathways that cause and advance HCC, as well as to discover possible indicators of prognosis and targets for treatment.

In recent years, with breakthroughs in cancer immunotherapy, increasing atten-tion has been given to the impact of characteristic gene mutations on the tumor mi-croenvironment [7]. The transcription factor KAISO (ZBTB33) can attach to both DNA and methylation marks on gene promoters or particular KAISO binding sites, enabling it to identify and control target genes [8, 9]. Interestingly, elevated levels of nuclear KAISO have been associated with increased invasiveness of cancer and a poorer prognosis [10, 11]. Furthermore, it has been observed that animals who do not possess Kaiso exhibit a state of immunity towards colorectal cancer [12]. Therefore, the accu-rate depiction and specific placement of KAISO are essential in controlling its normal physiological function and its contribution to the development of cancer.

IGFBP1, a gene found on chromosome 7q12.3, is made up of 4 exons and covers a length of 5173 base pairs. It is a crucial component of the IGF system [13, 14]. IGF-dependent activities have a significant impact on many tumor features, such as cell growth, movement, infiltration, and attachment [13]. Moreover, IGFBP1 plays a role in controlling the invasion of tumor immune cells in clear cell renal cell carcinoma, esophageal cancer, and metastatic melanoma [15]. Nevertheless, its function in HCC is still ambiguous. Therefore, it is crucial to examine the function and processes of IGFBP1 in the immunosuppression of HCC in order to discover novel therapeutic tar-gets for the treatment of HCC.

Certain features of HCC are frequently unknown and caused by unknown genetic backgrounds, the process of HCC growth and progression is complex [16, 17]. This highlights the critical importance of trustworthy biomarkers and the metabolic pro-cesses that lead to them [18].

KAISO predicted poor progression in pan-cancer

Using data from 33 different tumor types that showed increased levels of KAISO, we ran a survival study to determine the effect of KAISO on tumor survival (Fig. 1A). Many malignancies have Kaplan-Meier survival curves, including liver hepatocellular carcinoma (LIHC), kidney renal clear cell carcinoma (KIRC), breast invasive carcinoma (BRCA), brain lower grade glioma (LGG), sarcoma (SARC), and kidney chromophobe (KICH). The survival curves for disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI) are shown in Fig. 1B-G and Supplementary Figures S1-S3. The results show that survival outcomes in HCC patients are significantly impacted by increased KAISO expression.

Following an exhaustive survival analysis, it became evident that elevated levels of KAISO expression exert a detrimental influence on the outcomes of cancer patients, encompassing various tumor types, with a notable impact observed among those afflicted with HCC.

KAISO overexpressed in pan-cancer and promotes neutrophil infiltration

To validate KAISO expression across 33 tumor types, we conducted pan-cancer expression analysis. Our findings showed that KAISO was upregulated in 11 out of 33 tumor types, including HCC, compared to adjacent normal tissues (Fig. 2A).

Using bioinformatics tools, we expanded our study to examine the relationship between KAISO expression and immune cell infiltration in different kinds of tumors. Our analysis revealed a significant presence of neutrophils, highlighting their potential role in the tumor immune microenvironment (Fig. 2B-C).

KAISO acts as a transcription factor regulating the transcription of IGFBP1

Our study further investigated the regulatory mechanisms of KAISO in the HCC immune microenvironment, revealing that KAISO regulates IGFBP1 expression as a transcription factor (Fig. 3A, Table S1). IGFBP1, which binds to IGF I and II, modulates immune infiltration in various tumors [15]. Therefore, KAISO may induce neutrophil infiltration by regulating the expression of IGFBP1.

To further investigate how KAISO specifically regulates the transcription of IGFBP1, we first identified potential KAISO binding sites and sequences on the IGFBP1 promoter. The binding motif of KAISO with the IGFBP1 promoter is highlighted by the gene expression motif diagram (Fig. 3B). Through a dual-luciferase reporter assay, we found that KAISO can activate the IGFBP1 promoter region from − 1100 to + 1bp, thereby regulating the transcription of IGFBP1 (Fig. 3C). To validate this conclusion, we designed primers based on the potential binding sequences and conducted chromatin immunoprecipitation (ChIP) assays. The findings demonstrated that the targeted antibody successfully recognized and bound to the KAISO target protein. That KAISO can regulate IGFBP1 transcription by binding to certain promoter regions is demonstrated here. We proceeded by conducting an ESA, or electrophoretic mobility shift test. We utilized a DNA probe that was FAM-labeled and generated from the IGFBP1 promoter region in this experiment. We observed that in the reaction group with the mutant probe, the mutant probe could not bind to the specific transcription factor and thus did not form the band that the wild-type probe formed. This confirmed that our labeled probe contained the specific binding site for the transcription factor. In the reaction group with the KAISO antibody, the presence of the antigen-antibody complex, which has a larger molecular weight, resulted in a supershift band with a slower migration rate. The experimental results show that KAISO can bind to the IGFBP1 promoter region and control the transcription of this gene.

Publicly available data that we used to back up our findings demonstrated a strong association between KAISO and IGFBP1 expression (Figs. 3F-G). Additionally, we showed that suppressing KAISO expression significantly reduced IGFBP1 expression in a cellular setting (Fig. 3H).

KAISO induces HCC proliferation by activating G2/M and mitotic phases

To delve into the specific regulatory mechanisms by which KAISO induces HCC proliferation, we conducted pathway enrichment analysis. In HCC, the significantly enriched pathways include G2M and MITOTIC (Fig. 4A-C).

Therefore, our findings suggest that KAISO may influence tumor cell proliferation by regulating the cell cycle. Cell cycle experiments were conducted to provide further evidence of this. Compared to the control group, Huh7 cells whose KAISO expression was knocked down had a G2/M phase population that dropped from 20.9–11.2%. This suggests that Huh7 cell cycle progression is inhibited by silencing KAISO expression (Fig. 4D). An experiment for cell colony formation was performed to provide additional evidence of KAISO's effect on the cell cycle. The results showed that after knocking down the KAISO gene in Huh7 cells, both the size and number of colonies were significantly reduced (Fig. 4E). At the same time, we examined cell proliferation markers at the cellular level. When KAISO was knocked down, the expression of PCNA and Ki-67 also decreased correspondingly (Fig. 4F). In summary, KAISO regulates HCC proliferation by activating the G2/M and mitotic phases.

Inhibition of tumor growth by targeting KAISO through suppression of IGFBP1 expression and neutrophil infiltration

In our investigation into the mechanisms regulating tumor growth, to elucidate the impact of KAISO inhibition on tumor development, we utilized an in vivo tumor xenograft model. Subcutaneous tumor models in mice were established by injecting KAISO knockdown Huh7 cells and Huh7 cells with low expression of EGFP. PBMCs were intravenously injected into the mice to simulate the inflammatory microenvironment typically associated with tumor growth promotion. Tumor progression was monitored for four weeks, with regular measurements of tumor size conducted to generate growth curves for each experimental group. We found that knocking down KAISO slowed the tumor growth rate (Fig. 5A-B).

The collected tumor tissues underwent immunohistochemical (IHC) analysis to evaluate the expression levels of KAISO, IGFBP1, PCNA, and Ki67. Results showed that knocking down KAISO expression significantly reduced tumor growth (Fig. 5C), accompanied by decreased IGFBP1 expression and lower neutrophil infiltration in the tumor microenvironment. The proliferation markers PCNA and Ki67 were found to be significantly lower in tumors from KAISO knockdown mice compared to the control group, suggesting that these tumor tissues had a reduced proliferative ability, according to IHC analysis (Fig. 5D).

In conclusion, our research findings suggest that the inhibition of KAISO exerts potent anti-tumor effects by modulating IGFBP1 expression and attenuating neutrophil infiltration, ultimately impairing tumor growth.

Upregulation of KAISO expression in a mouse model of HCC

To further investigate the expression levels of KAISO in HCC, we utilized a diethylnitrosamine (DEN)-induced mouse model of in situ HCC [19]. Immunohistochemical analysis revealed a significant upregulation of KAISO, PCNA, and Ki67 expression as the liver tumors developed in these mice (Fig. 6). This observation suggests a potential association between KAISO overexpression and the progression of hepatocellular carcinoma in this murine model.

KAISO is upregulated in HCC tissues and leads to poor prognosis in patients

We used the HCCDB database to study the expression of KAISO in HCC tissues and how it affects patient prognosis. Compared to nearby normal tissues, HCC tissues exhibited increased levels of KAISO expression (Fig. 7A).

In 97 patients diagnosed with HCC and adjacent non-tumor tissues, we collected PCR samples at Qingdao University Affiliated Hospital. Figure 7B displayed a statistically significant increase in KAISO expression in HCC tissues (p < 0.001). Western blot analysis of specimens from 42 patients further validated elevated KAISO protein levels in HCC tumor tissues (Fig. 7C).

To further validate our findings, we used tissue microarrays and immunofluorescence to quantify KAISO expression. When comparing the tumor to the surrounding normal tissues, the results demonstrated a statistically significant difference (p = 1.78e-17, Fig. 7D). Patients with higher levels of KAISO expression had worse prognoses and shorter survival times, according to follow-up and survival curve analysis (Fig. 7E).

Genetic and epigenetic alterations in tumor cells are well known to significantly impact their malignant potential [20–23]. These changes influence proliferation, apoptosis, metastasis, the tumor immune microenvironment, and therapy response [24, 25]. Gene mutations, amplifications, or deletions can activate oncogenes or inactivate tumor suppressor genes, driving tumor initiation and progression [26, 27]. The tumor microenvironment consists of stromal cells, endothelial cells, and immune cells; the interactions between these cells and tumor cells are critical for tumor growth and treatment effectiveness [28–31].A number of cancers, including colorectal, breast, prostate, and pancreatic cancers, have been found to have elevated KAISO levels, according to prior studies. This provides more evidence that KAISO may have predictive value as a biomarker [32–34]. Nevertheless, there is a lack of study on KAISO in HCC. According to our research, KAISO is an important gene that controls the immune microenvironment and is involved in HCC formation. Overexpression of KAISO promotes tumor cell proliferation and invasion and influences the tumor microenvironment by modulating immune-related genes and pathways. Neutrophils, crucial for immune system homeostasis, are significantly affected by KAISO [35]. We found that KAISO adds to the intricate relationship between tumor cells and the immune system by inducing neutrophil infiltration into the tumor microenvironment. As a transcription factor, KAISO controls IGFBP1 transcription by binding to its promoter region. IGFBP1 regulates the tumor microenvironment and acts as a neutrophil marker [15]. Serum IGF-BPs control the turnover, transport, and tissue availability of IGF I and IGF-II. IGFBP1 controls the metabolic, survival, motility, and proliferation of cells via regulating the bioactivity of IGF-I. IGFBP1 has dual roles in cancer: inhibiting cell proliferation and motility in some malignancies [36] and, depending on the setting, boosting treatment resistance and tumor cell migration as an oncogene [37, 38]. Our findings align with these conclusions, showing that KAISO upregulation increases IGFBP1 expression, contributing to tumor growth and poor prognosis in patients.

Humanized immunodeficient mice, which have been implanted with components of the human immune system, are widely utilized in studies of human immunobiology. Studying the immune system's origins, maintenance, and operation falls within this category [39]. Introducing PBMCs into mice can mimic the human immune system's response, making the immune response of mice more similar to humans, thereby enhancing the reliability of disease models and the biological comparability [40]. In our study, inhibition of KAISO expression resulted in a corresponding decrease in tumor size in the mouse model. With any luck, this will lead to the realization of the dream of molecular therapy for malignancies by making KAISO a target for the treatment of HCC. This also provides important reference value for the subsequent development of drugs. In addition to the subcutaneous tumor model in mice, we also established an orthotopic tumor model in mice and assessed the expression of cell cycle-related biomarkers. This orthotopic model more closely mimics the natural tumor microenvironment and provides a valuable platform for studying tumor progression and therapeutic responses [41–44]. Our analysis of PCNA and Ki-67 expression, two cell cycle markers, allowed us to better understand the regulatory processes that drive tumor growth and to pinpoint possible areas of intervention [45–47].

The KAISO transcription factor has been implicated in a number of biological activities, according to prior studies. One way in which KAISO controls the cell cycle is via interacting with cyclins D1 and E1 [48]. Additionally, Kaiso is an important transcription factor in cancer cell proliferation and migration, and can enhance intestinal tumorigenesis in mice [49, 50]. This study further demonstrates that KAISO can regulate Ki-67 and PCNA to influence the cell cycle. Interfering with KAISO expression can inhibit the cell cycle progression of Huh7 cells, thereby achieving the goal of controlling tumor progression. Therefore, the regulation of tumor cells by KAISO is primarily achieved through the modulation of the tumor cell cycle.

A significant risk factor in the development and progression of HCC, increased expression of KAISO is linked to worse patient outcomes and disease advancement. Furthermore, KAISO's role as a transcription factor in regulating IGFBP1 expression underscores its involvement in promoting neutrophil infiltration within the tumor microenvironment. Conversely, inhibition of KAISO offers a promising avenue for controlling tumor progression. Moving forward, exploring KAISO as a potential therapeutic target for HCC warrants focused and tailored research efforts aimed at elucidating its mechanisms of action and therapeutic implications.

Patients and Animals

The following samples were obtained from HCC patients between 2014 and 2020: 97 pairs from Qingdao University Affiliated Hospital of tumor and adjacent non-tumor tissue; 183 pairs from Eastern Hepatobiliary Surgery Hospital, Naval Medical University. We also collected PBMCs, or peripheral blood mononuclear cells, from healthy people. Both universities' ethics boards gave their stamp of approval to the study because it followed all the guidelines laid down in the Declaration of Helsinki. A written informed consent form was requested of all subjects.

Viton Lever Biotechnology Co., Ltd. supplied the six-week-old male BALB/c and nude mice, which were housed in SPF-grade facilities. The Research on animals was approved by the Ethics Committee of Changsha Medical University (license number: 2023038), in accordance with protocols set forth by the National Institutes of Health (NIH) and the Animal Center at Changsha Medical University.

Mouse Models

Mice received weekly intraperitoneal DEN injections from one week old for four weeks, then raised until 10 months old and euthanized for tissue collection. For the subcutaneous tumor model, Huh7 cells were injected subcutaneously in nude mice, with PBMCs injected via the tail vein to create humanized immune-deficient mice. Before collecting tumor tissue from animals, we first anesthetize them with an intraperitoneal injection of pentobarbital sodium. This study is reported in accordance with ARRIVE guideline.

Immunohistochemistry

Samples were fixed in formalin, sectioned, and underwent antigen retrieval. Non-specific binding sites were blocked, followed by incubation with a primary antibody. After washing, a labeled secondary antibody was applied. Prior to being mounted for microscopic analysis, sections were treated with DAB stain, followed by hematoxylin counterstain, dehydrated, and cleaned in xylene.

Electrophoretic Mobility Shift Assay (EMSA)

DNA probes for KAISO binding sites on IGFBP1 were designed and synthesized. EMSA reactions mixed nuclear proteins with labeled probes in binding buffer, using PMSF and cold probes as competitors. Samples were loaded onto a 4% EMSA gel for electrophoresis, and chemiluminescent signals were detected with a Western blot imaging system.

Chromatin Immunoprecipitation (ChIP)

Following the cross-linking of cells with formaldehyde, the addition of glycine halted the reaction. After lysing the cells with protease inhibitor-containing SDS lysis solution, chromatin was fragmented by sonication. The target protein-chromatin fragments were bound using a particular antibody and Protein A/G agarose beads. Cross-links were reversed by heating, and impurities were removed with protease and RNase digestion. The purified DNA samples were then recovered for PCR analysis.

PBMCs Isolation

Lymphocyte separation medium was added to a 50 ml tube, and blood samples were layered on top. The cell pellet was spun again at 2000 rpm after being resuspended in 1x PBS. To prepare the PBMCs, the cells were rinsed twice with 1x PBS, the RBCs were lysed, and the rest of the cells were rinsed twice more. Collecting whole blood cells from individuals during health check-ups does not pose any additional economic or health burden on those undergoing the examination. The procedure was approved by the participants, and informed consent forms were signed.

Quantitative RT-PCR

Total RNA was extracted from cells or tissues using an RNA extraction reagent such as Trizol. The extracted RNA was then used as a template for cDNA synthesis, which was performed by adding reverse transcriptase, random primers or specific primers, dNTPs, and reverse transcription buffer. Using the synthesized cDNA as a template, specific primers for KAISO were designed. The PCR reaction mixture included Taq polymerase, dNTPs, PCR buffer, and SYBR Green. PCR amplification was conducted, and the fluorescence signals were quantitatively analyzed using software provided with the PCR machine. The statistical analysis was carried out on the determined relative expression levels of the target gene.

Western Blot

A BCA Protein Assay Kit (P0012, Beyotime) was used to measure the proteins that were liberated after cell disruption. After denaturing the proteins, loading buffer was added to the samples, and they were finally loaded onto an SDS-PAGE gel. After the proteins were electrophoresed, they were moved to a membrane. Initial steps included blocking the membrane and treating it with GAPDH, KAISO, Ki-67, PCNA, and IGFBP1 primary antibodies. Subsequently, enzyme-labeled secondary antibodies were added. The membrane underwent a washing process, followed by incubation with a chemiluminescent substrate. The resulting protein bands were then seen using the Tanon imaging system.

Cell Colony Formation Assay

Cells in the logarithmic growth phase were harvested, enzymatically digested, and quantified. A bilayer agar was created, consisting of a solidified agar layer at the bottom and a layer of cell suspension on top. Huh7 cells were introduced onto the upper layer of agar and left to incubate until colonies developed. The colonies were treated with formaldehyde, dyed with crystal violet, and captured in photographs.

Cell Cycle Assay

Cells were collected at specific time points to ensure an adequate number of cells for subsequent analysis. The cells were fixed using cold ethanol (70%) or another fixative to preserve cell morphology and DNA structure. Fixed cells were then mixed with propidium iodide (PI) staining solution to stain the DNA, facilitating analysis by flow cytometry or observation under a fluorescence microscope. The DNA content of the cells was detected using a flow cytometer, which distinguishes cells at different stages of the cell cycle based on fluorescence intensity. Using the flow cytometer data, we generated a graph showing the distribution of the cell cycle by comparing the relative quantity of cells in each phase (G1, S, G2, M).

Multiplex Immunofluorescence

Xylene and ethanol were used for deparaffinization of the tissue slices. After that, the antigen was extracted by heating in an EDTA buffer in a microwave. Inhibiting endogenous peroxidase activity and preventing non-specific binding were both achieved with the use of BSA. Primary antibodies were applied to the sections, and then HRP-conjugated secondary antibodies were used. The nuclei were stained with DAPI, and CY3-TSA and FITC-TSA were used in the detection process. Using a fluorescent microscope, the sections were examined and photographed.

Statistical Analysis

The FindClusters tool was used to discover cell clusters at a resolution of 0.5. Marker genes from the Human Cell Atlas were then used to label the clusters. Gene Set Enrichment Analysis (GSEA) was used to assess tumor and surrounding tissue data given by the Cancer Genome Atlas (TCGA). The statistical experiments were carried out in R (v4.2.3) and included t-tests, log-rank tests for survival (Progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), disease interval survival (DIS)), and stepwise Cox regression. P < 0.05 was set as the significant level. The data is shown as the mean value elevated or lowered relative to the standard deviation.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (grant no. 82303364), the Natural Science Foundation of Hunan Province (grant no. 2023JJ40089), and the Education Department of Hunan Provincial (grant no. 22B0895).

Author contributions

Yang TW, Pang YQ, Wang HJ, and Zhou J contributed to data collection and analysis. Yang TW, Li Q, and Zhou J contributed to study design. Yang TW, Wang YT, and Li Q assisted in preparing the manuscript. Yang TW, Pang YQ supervised the study. All authors have thoroughly reviewed and approved the final manuscript.

Data availability statement

Data utilized and analyzed in this study can be obtained from the corresponding author upon reasonable request.

Conflict of interest

The authors declare that they have no competing interests

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No competing interests reported.

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Reviewers agreed at journal
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KAISO Promotes Poor Prognosis in Hepatocellular Carcinoma Patients by Enhancing Neutrophil Infiltration via IGFBP1

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Version 1

Abstract

Figures

Introduction

Results

KAISO predicted poor progression in pan-cancer

KAISO overexpressed in pan-cancer and promotes neutrophil infiltration

KAISO acts as a transcription factor regulating the transcription of IGFBP1

KAISO induces HCC proliferation by activating G2/M and mitotic phases

Inhibition of tumor growth by targeting KAISO through suppression of IGFBP1 expression and neutrophil infiltration

Upregulation of KAISO expression in a mouse model of HCC

KAISO is upregulated in HCC tissues and leads to poor prognosis in patients

Discussion

Methods

Patients and Animals

Mouse Models

Immunohistochemistry

Electrophoretic Mobility Shift Assay (EMSA)

Chromatin Immunoprecipitation (ChIP)

PBMCs Isolation

Quantitative RT-PCR

Western Blot

Cell Colony Formation Assay

Cell Cycle Assay

Multiplex Immunofluorescence

Statistical Analysis

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1