Pescadillo ribosomal biogenesis factor 1 and programmed death-ligand 1 in gastric and head and neck squamous cell carcinoma

Abstract

BACKGROUND

Gastric cancer (GC) and head and neck squamous cell carcinoma (HNSCC) are common malignancies with high morbidity and mortality rates. Traditional treatments often yield limited efficacy, especially in advanced cases. Recent advancements in immunotherapy, particularly immune checkpoint inhibitors targeting programmed death-ligand 1 (PD-L1), have shown promise. However, the expression and interaction of pescadillo ribosomal biogenesis factor 1 (PES1) and PD-L1 in these cancers remain unclear. Understanding their roles could provide new insights into tumor biology and improve therapeutic strategies.

AIM

To investigate the expression levels of PES1 and PD-L1 in tumor tissues of patients with GC and HNSCC.

METHODS

A total of 58 cases of GC and HNSCC undergoing surgical resection were selected from January 2022 to January 2024. Paraffin specimens of GC and HNSCC tissues were taken from the patients, and the sections were subjected to staining with immunohistochemistry and hematoxylin-eosin staining, and the protein expression of PES1 and PD-L1 was observed microscopically.

RESULTS

Among 58 GC and HNSCC tissues, 30 cases were positive and 28 cases were negative for PES1 expression, and 34 cases were positive and 24 cases were negative for PD-L1 expression. The positive expression rates of PES1 and PD-L1 were 51.72% and 58.62%, respectively. PES1 expression was correlated with the TNM stage, lymph node metastasis, and the depth of infiltration (P < 0.05), and PD-L1 expression was correlated with the differentiation degree, lymph node metastasis, and infiltration depth (P < 0.05).

CONCLUSION

PES1 and PD-L1 were positively expressed in GC and HNSCC tissues and correlated with clinical features. They may serve as potential biomarkers for immune-targeted therapies.

Key Words: Pescadillo ribosomal biogenesis factor 1; Programmed death-ligand 1; Gastric cancer; Head and neck squamous cell carcinoma; Expression level

Core Tip: This study investigated the expression of pescadillo ribosomal biogenesis factor 1 (PES1) and programmed death-ligand 1 (PD-L1) in tumor tissues from patients with gastric cancer and head and neck squamous cell carcinoma. We found that both proteins exhibited significant positive expression rates (51.72% for PES1 and 58.62% for PD-L1) and were associated with clinical features such as TNM stage and lymph node metastasis. Our results suggest that PES1 and PD-L1 may play critical roles in tumor progression and immune escape, potentially serving as biomarkers for immune-targeted therapies.

INTRODUCTION

Gastric cancer (GC) and head and neck squamous cell carcinoma (HNSCC) are common malignant tumors that pose a grave menace to human health worldwide[1,2]. GC is ranked fifth in tumor frequency across the globe and is the fifth-leading cause of cancer-related deaths, especially in China where its incidence and mortality rates remain high[3]. This scenario not only presents a significant danger to the well-being of patients but also imposes a substantial strain on the societal public health infrastructure. The high morbidity and mortality of GC requires continuous innovation and breakthroughs in treatment strategies, especially for patients with advanced and/or recurrent disease in which existing treatments, such as chemotherapy and radiotherapy, have limited efficacy.

Meanwhile, head and neck tumors are a part of the most common and widespread malignant tumors worldwide. HNSCC is the most frequently occurring head and neck tumor. The effectiveness of conventional treatments, especially in patients with advanced or recurrent disease, is still limited, and there is a pressing necessity to identify novel treatment approaches, particularly for patients who are resistant to chemotherapy or experience recurrence[4].

In recent years, with the rise of immunotherapy, especially the clinical application of immune checkpoint inhibitors, new hope has been brought to tumor treatment. Among them, programmed death-ligand 1 (PD-L1), as an important immune checkpoint molecule, is strongly expressed in a spectrum of tumor tissues and inhibits T cell activity by binding to programmed death 1 (PD-1), leading to tumor immune escape[5,6]. The PD-L1/PD-1 pathway is a cornerstone of regulating immune response and maintaining immune tolerance, and its inhibitors can block this pathway and restore T cell activity, thus enhancing immune response and inhibiting tumor growth.

PD-L1 has been demonstrated to be highly elevated in GC and HNSCC, with its expression level being significantly in line with clinicopathological features and patient outcomes. In GC, the expression of PD-L1 was closely linked to the depth of infiltration of tumor cells, the degree of differentiation, and the survival of patients. In HNSCC, PD-L1 expression was also closely in line with clinical stage. Additionally, patients with PD-L1 positivity had a higher likelihood of deriving benefits from anti-PD-1/PD-L1 treatment[7-10]. Although the important role of PD-L1 in tumor immunotherapy has been widely recognized, much remains unknown about its expression mechanism and interaction with other immune molecules.

Pescadillo ribosomal biogenesis factor 1 (PES1), as a new biomarker, has not yet been intensively investigated in terms of its expression in tumor tissues and its relationship with PD-L1[11]. Therefore, the purpose of this study was to detect the expression of PES1 and PD-L1 in tumor tissues of patients with GC and HNSCC, to explore the differences in their expression in different pathological grades and clinical stages, to provide theoretical and experimental bases for the immune-targeted therapy of GC and HNSCC, and to provide new ideas for the future immunotherapeutic strategies.

MATERIALS AND METHODS

Patient selection

A total of 58 cases of GC and HNSCC admitted to our hospital for surgical resection between January 2022 and January 2024 were selected, all of which were definitively diagnosed as GC or HNSCC by pathology. Among them, there were 37 male patients and 21 female patients. The age range was 39 to 72 years with a mean of 52.33 ± 8.29 years. Surgical resection specimens were used for analysis, and other relevant clinical data were taken from patients’ pathology reports and medical records. The utilization of the specimens for clinical purposes in this study was examined and sanctioned by the hospital’s Ethics Committee.

Inclusion criteria were: (1) Patients and their families have signed informed consent; (2) Patients have not received radiotherapy, chemotherapy, or immunotherapy for tumors; and (3) All specimens were fixed in 40 g/L formaldehyde and embedded in paraffin within 60 min after removal from the body, and the thickness of the tissue sections was 4 μm. Exclusion criteria were: (1) Patients with metastatic tumors or recurrent tumors; (2) Patients with severe consciousness disorders; (3) Patients with combined cardiac, pulmonary, and renal function abnormalities; (4) Patients with pathological conditions; and (5) Patients with incomplete pathological and clinical data. The research was endorsed by the hospital’s Ethics Committee, and all participants provided informed consent.

Reagents and instruments

Reagents: (1) PES1 antibody. Select a highly specific and sensitive PES1 monoclonal antibody for detecting PES1 expression in tumor tissues; (2) PD-L1 antibody. Select a highly specific and sensitive PD-L1 monoclonal antibody for detecting PD-L1 expression in tumor tissues; (3) Immunohistochemistry staining kit. Contains all necessary stains such as hematoxylin, eosin, etc, reagents, antibody diluent, fixative, dehydrating agent, transparency agent, etc., for immunohistochemical staining of tumor tissues; (4) PBS. Use to wash the samples and remove excess antibodies and stains; (5) Sealing agent. Use to seal the samples, protect the samples, and prevent discoloration; and (6) Pipette. Use for the precise addition of samples, such as antibodies and staining solutions.

Instruments: (1) Microscope. For observing the results of immunohistochemical staining and evaluating the expression of PES1 and PD-L1 in tumor tissues; (2) Thermostatic incubator. For temperature control during fixation, dehydration, transparency, and staining of the samples; (3) Paraffin slicer. For cutting paraffin-embedded tumor tissue blocks into thin slices; and (4) Tissue dehydrator. For the gradual transition of the tissue samples from a watery state to solvents such as alcohol and xylene to facilitate paraffin embedding.

Sample collection

Representative tumor tissues from patients with GC and HNSCC were obtained under sterile conditions by surgical resection or biopsy. Surgically resected tumor tissues were immediately placed in formalin fixative, and the fixation time was usually 24-48 h. After fixation, the tissue samples were processed into paraffin blocks through dehydration, clarification, wax immersion, and embedding procedures and stored in a 4 °C refrigerator for a long time. Before immunohistochemical testing, 4-6 μm thick slices were cut out from the paraffin blocks, attached to anti-detachment slides, and dried at 60 °C.

Immunohistochemical testing

Sections were immersed twice in xylene for 10 min each time to remove paraffin and then sequentially hydrated by passing through a gradient of 1000 g/L, 950 g/L, 900 g/L, 800 g/L, and 700 g/L ethanol for 5 min each time. Finally, the sections were washed with distilled water to remove any residual ethanol.

Antigen repair

Sections were positioned in citrate buffer (pH 6.0) and thermally repaired with antigen using a microwave oven or an autoclave to increase antigen exposure and antibody binding efficiency. After repair was completed, the sections were allowed to cool to ambient temperature and subsequently rinsed with PBS three times, each for a duration of 5 min.

Sections were treated with 30 g/L hydrogen peroxide solution and incubated at room temperature for 10 min to quench endogenous peroxidase activity, followed by rinsing with PBS three times, each for 5 min. Sections were closed using 50 g/L goat serum or bovine serum albumin and incubated at ambient temperature for a duration of 30 min to minimize nonspecific binding.

Appropriate dilution ratios of PES1 and PD-L1 specific primary antibodies were added dropwise to the sections, ensuring that the antibodies covered the tissue area evenly. The sections were placed in a wet box and incubated overnight at 4 °C or for 1-2 h at ambient temperature. The sections were washed with PBS three times, each time for 5 min to remove the unbound primary antibody.

Horseradish peroxidase-labeled secondary antibody was added dropwise on the sections and incubated at room temperature for 30 min to allow the reaction to proceed. The sections were washed with PBS 3 times for 5 min each time. Freshly prepared DAB staining solution was added by drops on the sections. The staining was observed under the microscope and immediately rinsed with distilled water to terminate the staining when a brown precipitate appeared.

Sections were re-stained using hematoxylin to color the nuclei, followed by hydrochloric acid alcohol differentiation and ammonia return to blue. Sections were subjected to graded ethanol dehydration (700 g/L, 800 g/L, 900 g/L, 950 g/L, 1000 g/L) and xylene transparency. Neutral gum was used to seal the sections, ensuring that the sections were flat and free of air bubbles. The slices were observed under a microscope, and the staining of PES1 and PD-L1 was recorded. The positive expression rate was evaluated according to the staining intensity and distribution range.

Observation indicators

(1) Expression of PES1 and PD-L1 in GC and HNSCC tissues; and (2) Association between PES1 and PD-L1 expression levels and the clinical characteristics of GC and HNSCC.

Statistical analysis

Statistical analysis was conducted using SPSS 25.0. Measurement data that followed a normal distribution were presented as mean ± SD. Comparisons between groups were made using the t test. Counting data were represented as n (%), with the χ² test employed for analysis. The immunohistochemical staining expression of PES1 and PD-L1 proteins in GC and HNSCC tissues was evaluated using Fisher’s discriminant method (Fisher’s test). Statistical significance was set at P < 0.05.

RESULTS

Expression of PES1 and PD-L1 in GC and HNSCC tissues

Among 58 cases of GC and HNSCC, 30 cases were positive and 28 cases were negative for PES1 expression. In addition, 34 cases were positive and 24 cases were negative for PD-L1 expression. The positive expression rates of PES1 and PD-L1 were 51.72% and 58.62%, respectively (Table 1).

Table 1 Expression of pescadillo ribosomal biogenesis factor 1 and programmed death-ligand 1 in gastric cancer and head and neck squamous cell cancer tissues.

	Negatives	Positive
PES1	28 (48.28)	30 (51.72)
PD-L1	24 (41.38)	34 (58.62)

Data are presented as n (%). PES1: Pescadillo ribosomal biogenesis factor 1; PD-L1: Programmed death-ligand 1.

Association between PES1 and PD-L1 expression levels and the clinical characteristics of GC and HNSCC

PES1 expression correlated with TNM stage, lymph node metastasis, and infiltration depth (P < 0.05), while it was not correlated with gender, age, degree of differentiation, tumor diameter, and diabetes mellitus (P > 0.05). PD-L1 expression was correlated with degree of differentiation, lymph node metastasis, and infiltration depth (P < 0.05) but was not correlated with gender, age, TNM stage, tumor diameter, and diabetes (P > 0.05) (Tables 2 and 3).

Table 2 Relationship between pescadillo ribosomal biogenesis factor 1 expression and clinical features of gastric cancer and head and neck squamous cell carcinoma.

Diagnostic trait	n	PES1		χ2	P value
		Negative (n = 28)	Positive (n = 30)
Sex				0.222	0.637
Male	37	17 (45.95)	20 (54.05)
Women	21	11 (52.38)	10 (47.62)
Age (years)				0.210	0.647
< 60	41	19 (46.34)	22 (53.66)
≥ 60	17	9 (52.94)	8 (47.06)
Degree of differentiation				0.672	0.412
Poorly differentiated	32	17 (53.13)	15 (46.88)
Moderately to well differentiated	26	11 (42.31)	15 (57.69)
TNM staging				8.312	0.004
I-II	28	19 (67.86)	9 (32.14)
III	30	9 (30.00)	21 (70.00)
Lymphatic node transfer				5.287	0.021
Yes	39	15 (38.46)	24 (61.54)
No	19	13 (68.42)	6 (31.58)
Infiltration depth				6.846	0.009
T1/T2	25	17 (68.00)	8 (32.00)
T3/T4	33	11 (33.33)	22 (66.67)
Tumor diameter (cm)				0.485	0.486
< 5	41	21 (51.22)	20 (48.78)
≥ 5	17	7 (41.18)	10 (58.82)
Diabetes				0.021	0.885
Yes	15	7 (46.67)	8 (53.33)
No	43	21 (48.84)	22 (51.16)

Data are presented as n (%). PES1: Pescadillo ribosomal biogenesis factor 1.

Table 3 Relationship between programmed death-ligand 1 expression and clinical characteristics of gastric cancer and head and neck squamous cell carcinoma.

Diagnostic trait	n	PD-L1		χ2	P value
		Negative (n = 24)	Positive (n = 34)
Sex				0.879	0.349
Male	37	17 (45.95)	20 (54.05)
Women	21	7 (33.33)	14 (66.67)
Age (years)				0.367	0.545
< 60	41	18 (43.90)	23 (56.10)
≥ 60	17	6 (35.29)	11 (64.71)
Degree of differentiation				6.508	0.011
Poorly differentiated	32	18 (56.25)	14 (43.75)
Moderately to well differentiated	26	6 (23.08)	20 (76.92)
TNM staging				0.049	0.825
I-II	28	12 (42.86)	16 (57.14)
III	30	12 (40.00)	18 (60.00)
Lymphatic node transfer				5.525	0.019
Yes	39	12 (30.77)	27 (69.23)
No	19	12 (63.16)	7 (36.84)
Infiltration depth				9.269	0.002
T1/T2	25	16 (64.00)	9 (36.00)
T3/T4	33	8 (24.24)	25 (75.76)
Tumor diameter (cm)				0.000	0.984
< 5	41	17 (41.46)	24 (58.54)
≥ 5	17	7 (41.18)	10 (58.82)
Diabetes				1.192	0.275
Yes	15	8 (53.33)	7 (46.67)
No	43	16 (37.21)	27 (62.79)

Data are presented as n (%). PD-L1: Programmed death-ligand 1.

DISCUSSION

GC ranks as one of the foremost causes of cancer-related fatalities on a global scale[12,13]. The majority of patients with GC are in advanced stages at the time of diagnosis and have shorter survival times and poor overall quality of life[14]. Traditional treatments include surgery, radiotherapy, and chemotherapy, but for advanced GC the cure rate of surgery is low, and the efficacy of radiotherapy is poor. Over the past few years, the development of immunotherapy and the achievement of certain clinical efficacy have made it a new treatment mode, in which immune checkpoint inhibitors play an important role[15-17].

PES1 is a nucleolin protein and has gradually been paid attention to in recent years[18]. It has been noted that in GC cell lines (AGS and N87) inhibition of PES1 expression leads to cell cycle arrest in the G1 phase, a finding that implies that low expression levels of PES1 play a part in inhibiting cell proliferation[19]. However, PES1 has not received sufficient attention within the field of GC research, and its specific significance in the clinical diagnosis and prognostic assessment of GC remains unclear.

HNSCC is a routine malignant tumor that is highly heterogeneous[20]. Although classical tumor TNM staging has been widely used for clinical diagnosis and to assist in clinical treatment decisions, TNM staging is based only on the tumor itself and fails to accurately predict the projection of patients with HNSCC. More and more studies have shown that various immune cells in the tumor microenvironment, especially B cells, contribute significantly to tumor progression and prediction of outcomes[21-24]. However, the presentation of PES1 in HNSCC and its relationship with immune infiltration have not been clarified.

In view of this, GC and HNSCC were chosen as the subjects of this study, covering two common malignant tumors, thus expanding the scope of the study. For the detection method, we used monoclonal antibodies with high specificity and sensitivity for immunohistochemical detection, which improved the accuracy of the results. In addition, this study explored the relationship between PES1 and PD-L1 for the first time, which provided new ideas for future research on the synergistic role of both in tumor immune escape.

In this study, 58 GC and HNSCC tissues were examined, and the experiments established that PES1 and PD-L1 had positive expression rates of 51.72% and 58.62%, respectively, in both tumor tissues. This finding suggests that PES1 and PD-L1 may contribute significantly to the development and progression of GC and HNSCC. Specifically, the expression level of PES1 may change with the increase of tumor malignancy and may be involved in the process of tumor invasion and metastasis. It has been shown that the high expression of PES1 in tumor tissues may be closely related to tumor development and progression, and as a potential biomarker for some tumors, it serves the purpose of early diagnosis, prognostic assessment, and monitoring of therapeutic efficacy[25-27].

PES1 is an important biomarker, and its aberrant expression has been in line with the onset and progression of a variety of cancers. In some tumors, high expression of PES1 tends to predict a more malignant, aggressive, and metastatic tumor. Therefore, PES1 may be a powerful indicator for assessing the prognosis of tumor patients. In addition, the expression of PES1 has certain specificity in the tissues of different tumors and makes its application in individualized therapy a great potential. In this study, we found that PES1 was contingent upon high expression rates in tumor tissues of both GC and HNSCC, a result that further supports the value of PES1 as a potential biomarker.

In terms of PD-L1, the findings of this research indicated a substantial association between its expression and the degree of differentiation, lymph node metastasis, and depth of infiltration of the tumor (P < 0.05). This finding is in accordance with earlier studies and suggests that PD-L1 is vital to tumor immune escape. PD-L1 inhibits T cell activation and proliferation by binding to PD-1 on the surface of T cells, which in turn helps tumor cells to dodge surveillance and clearance by the immune system[28-30]. This immune escape mechanism enables tumors to continue to grow under the suppression of the immune system and exhibits strong invasiveness and metastatic ability. Therefore, high PD-L1 expression is usually highly tied to malignant biological behaviors of tumors, such as high tumor aggressiveness, high metastatic capacity, and poor prognosis.

However, unlike PES1, PD-L1 expression showed no significant association with TNM staging (P > 0.05). This result suggests that more complex factors may need to be considered when assessing the clinical significance of PD-L1. For example, the tumor microenvironment, immune cell infiltration, and other immune escape mechanisms may have an impact on the expression level of PD-L1. Therefore, the independence of PD-L1 expression from TNM staging also suggests that relying solely on PD-L1 as a prognostic marker may have certain limitations and that multiple factors must be combined to more accurately predict patient prognosis.

In addition, this study also found that PD-L1 expression was independent of patient gender, age, tumor diameter, and diabetes mellitus status (P > 0.05), which further supported the exact function of PD-L1 in tumor immunomodulation. These results suggest that the important role played by PD-L1 in the immune escape process is not affected by these common clinical features and makes its potential and application value in tumor immunotherapy even more prominent.

Combined with the observations made in this research, we can speculate that PES1 and PD-L1 may serve as potential biomarkers for GC and HNSCC and that their high expression may be intertwined with tumor invasiveness, metastatic ability, and poor prognosis. Recent studies have shown that immune checkpoint molecules play a key role in tumor immune escape. PD-L1, as an important immune checkpoint molecule, inhibits the activation and proliferation of T cells by binding to PD-1 on the surface of T cells, which in turn helps tumor cells to evade the surveillance and clearance of the immune system.

The abnormal expression of PES1 in tumors may be related to immune regulation. In this study, we found that PES1 and PD-L1 had certain positive expression rates in GC and HNSCC tissues, and the expression of PES1 was related to TNM staging, lymph node metastasis and infiltration depth, while the expression of PD-L1 was related to differentiation degree, lymph node metastasis, and infiltration depth. This suggests that PES1 and PD-L1 may interact with each other and participate in the immune escape process of the tumor. This finding provides new ideas and directions for clinical selection of appropriate therapies, especially in the context of immunotherapy, and PES1 and PD-L1 may provide valuable references for individualized treatment. However, more large-sample studies and in-depth mechanistic studies are needed to verify this hypothesis.

Notably, although this study revealed the expression of PES1 and PD-L1 in GC and HNSCC and their relationship with clinical features, their interaction has not been directly explored yet. In view of this, future studies could further explore the interaction mechanism between PES1 and PD-L1. From the theoretical analysis, PES1 may regulate the expression level of PD-L1 by affecting the signaling pathways in tumor cells and thus regulating the expression level of PD-L1. For example, PES1 may promote the transcription of the PD-L1 gene by affecting the activity of certain transcription factors, thereby increasing the protein expression of PD-L1. In addition, PES1 may also indirectly affect the function of PD-L1 by influencing immune cell infiltration in the tumor microenvironment. For example, high expression of PES1 may attract more immune-suppressive cells to infiltrate into tumor tissues, and these immune-suppressive cells may enhance the immunosuppressive function of PD-L1 by secreting cytokines, thereby promoting immune escape from the tumor.

An in-depth study of the interaction between PES1 and PD-L1 will not only help to further reveal the mechanism of tumor immune escape but may also provide an important theoretical basis for the development of new tumor immunotherapy strategies. For example, if we can find the key targets to block the interaction between PES1 and PD-L1, it may provide new ideas for combination immunotherapy, thus improving the effectiveness of tumor treatment. In addition, exploring whether PES1 and PD-L1 can be used as biomarkers for joint prediction of tumor prognosis is also an important direction for future research. By gaining a deeper understanding of the roles of these two in tumor immunomodulation, we may be able to develop more precise and effective therapeutic strategies to further enhance the efficacy of tumor therapy and the quality of patient survival.

CONCLUSION

This study provided valuable information on the articulation of PES1 and PD-L1 in GC and HNSCC and their clinical significance. However, the present study has some limitations, such as a small sample size, which limits the generalizability and reliability of the findings. In addition, the current study only conducted univariate analysis and did not consider the effects of potential confounders such as age and gender. Therefore, future studies need to further expand the sample size and employ multifactorial regression analyses to more comprehensively reveal the roles of PES1 and PD-L1 in tumor biology to more accurately assess the relationship between their expression and the clinical features of GC and HNSCC.