Research ArticleExperimental Studies
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Lymphocyte Antigen 6 Family Member D (LY6D) Affects Stem Cell Phenotype and Progression of Pancreatic Adenocarcinoma

SHUMPEI OKIMURA, NAOHIRO NISHIDA, HIDEKAZU TAKAHASHI, YUHKI YOKOYAMA, HIROYUKI YAMAMOTO, ATSUSHI HAMABE, TAKAYUKI OGINO, NORIKATSU MIYOSHI, HIDENORI TAKAHASHI, MAMORU UEMURA, SHOGO KOBAYASHI, MASAKI MORI, YUICHIRO DOKI, HIDETOSHI EGUCHI and HIROFUMI YAMAMOTO
Anticancer Research November 2024, 44 (11) 4737-4749; DOI: https://doi.org/10.21873/anticanres.17300

Abstract

Background/Aim: The membrane-bound protein lymphocyte antigen 6 family member D (LY6D), a marker of early B cell lineage is reportedly expressed in several human malignancies and has been implicated in cancer stemness. However, its expression and role in cancer stemness remain largely unexplored in pancreatic ductal adenocarcinoma (PDAC). The aim of this study was to clarify the role of LY6D in PDAC. Materials and Methods: We conducted functional analysis of LY6D to evaluate its impact on the malignant features of PDAC cells in vitro. Using our in-house developed stem cell separation technique, which isolates cells with low proteasome activity and CD44 v9 cell surface marker for cancer stem cells, we performed sphere formation and chemosensitivity tests and tumor formation assay in mice, through knockdown of LY6D expression. Immuno-histopathological analysis was also conducted to reveal the clinical significance of LY6D in PDAC. Results: In vitro functional assays demonstrated that LY6D was critically involved in promoting the cancer malignant phenotype, including increased invasive ability, drug resistance, migration capacity, and cancer stemness. Immunohistopathological analysis revealed that high LY6D expression levels were associated with high recurrence rates and poorer prognosis in PDAC. Conclusion: Our study showed that LY6D is a novel prognostic indicator and plays a key role in regulation of cancer stemness in PDAC.

Key Words:

Pancreatic ductal adenocarcinoma (PDAC) is among the most life-threatening cancers, with over 400,000 new cases emerging every year worldwide (1). Despite advances in treatment, PDAC remains a leading cause of cancer-related death in many industrialized countries (2). Its poor prognosis is partly due to the fact that approximately 90% of tumors are diagnosed at an advanced stage where the tumor has already spread beyond the pancreas with systemic metastases present in over 50% of patients (3, 4). PDAC treatment requires a multidisciplinary approach, including both chemotherapy and radiotherapy. Even with such treatment, patients often experience treatment resistance and cancer recurrence, largely due to the existence of cancer stem-like cells (CSCs) (5).

CSCs are defined as cancer cells with a self-renewal capacity and multilineage potency. They constitute a small population within the bulk of the tumor and play critical roles in tumorigenicity, cancer progression, metastasis and recurrence (6, 7). The CSC model has attracted significant attention because it offers an explanation for the clinical observation that even when cancer treatments initially seem to eliminate cancer cells, the cancer can later reoccur.

LY6D is a membrane-bound protein that attaches to the cell surface through a C-terminal glycosylphosphatidylinositol (GPI) anchor (8). It exhibits lineage-specific expression, and is thus commonly used as a surface marker to identify leukocyte subsets, however, its physiological function in cancer progression is poorly understood. Genes of the Ly6 family are located on chromosome 8q24 alongside c-Myc. Somatic copy number gain in the 8q region is the most common type of copy number gain in several types of cancer (9, 10). Within this gene family, LY6D is located in the 8q24.3 region, which is frequently amplified in various types of human malignancies. Recent studies show that LY6D is associated with distant metastasis in estrogen receptor (ER) -positive breast cancer and poor prognosis in lung cancer (11, 12). Moreover, it has been suggested that LY6D may be involved in cancer stemness in lung cancer and laryngeal cancer (13).

Currently, only limited evidence links LY6D to pancreatic cancer. To explore the role of LY6D in cancer stemness, we used the ornithine decarboxylase (ODC) degron system to identify cells with characteristics resembling cancer stem cells. In this system, 26S proteasome recognizes the degron sequence, leading to degradation of the targeted protein. Under a microscope, cancer cells with low-proteasome-activity are visualized using stable expression of green fluorescence (ZsGreen) fused to the ODC degron, albeit at a low level. These cells are likely to display CSC-like traits, including strong sphere-forming ability, increased tumorigenicity, and elevated expression of CSC markers (14).

In the present study, to explore how LY6D is involved in stemness, we used cells characterized by low proteasome activity and CD44v9 positivity, which were flow-sorted from Panc-1 cells with higher stemness using the ODC degron system. Our aim was to investigate the possible role of LY6D in cancer stemness and its clinical significance as a prognostic factor in PDAC. We also explored the therapeutic implications of targeting this gene in PDAC.

Materials and Methods

Cell lines and culture conditions. The cell culture and quality maintenance techniques have been previously described (15). The human pancreatic cancer cell line Panc-1 was obtained from American Type Culture Collection (Manassas, VA, USA). Cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, 08456–36; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified incubator with 5% CO2. All experiments were performed with cells passaged <8 times.

Transduction of the degron reporter into pancreatic cancer cells. The low-proteasome (LP) activity cell isolation system was established by engineering cells that stably expressed ZsGreen fused to the carboxyl terminal degron of ornithine decarboxylase (ODC), as previously described (16). The degron sequence of ODC is directly degraded by proteasomes. Consequently, cells with low proteasome activity accumulate the fluorescent fusion protein and can be detected by fluorescent microscopy or flow cytometry (FITC channel).

Flow cytometry. Cell sorting was performed using the FACSAria II (BD Biosciences, Franklin Lakes, NJ, USA), and analysis was conducted with FACSDiva software (BD Biosciences). Cells were washed with PBS containing 2% FBS and then incubated with the primary antibody, anti-CD44v9 (Cosmo Bio, Tokyo, Japan), at 4°C for 20 min. To detect CD44v9, PE mouse anti-rat IgG2a (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was used as the secondary antibody. Degron+, cells were detected using Cell Sorter SH800Z (Sony Biotechnology Inc., San Jose, CA, USA). The cell population with high fluorescence intensity was collected by the EGFP channel as we previously described (14, 17).

Clinical tissue samples. PDAC tissue samples (n=75) were collected during surgeries performed between 2007-2012, at the Department of Gastroenterological Surgery, Osaka University. All patients had clear diagnoses with PDAC, according to the clinicopathological criteria described by the Japanese Society for pancreatic cancer. Samples were fixed in buffered formalin at 4°C overnight, processed through graded ethanol solutions, and embedded in paraffin. The specimens were appropriately used, with approval by the Ethics Committee at the Graduate School of Medicine, Osaka University.

Construction of the LY6D-shRNA lentivirus. We used two short hairpin RNA (shRNA) sequences specifically targeting LY6D (GeneBank ID:8581; 5′-ATCTGGTGAAGAAGGACTGTG-3′ and 5′-CCAGCAACTGCAAGCATTCTG-3′). Control cells were generated by transfecting cell with empty vector. The LY6D gene was cloned into the enhanced green fluorescent protein (eGFP) containing pReceiver-Lv193x lentivector (iGene Biotechnology Co., Ltd., Columbia, MD, USA) using FastDigest KpnI (cat. no. FD0524) and XhoI (cat. no. FD0694) restriction endonucleases (both Thermo Fisher Scientific, Inc., Waltham, MA, USA) to produce plasmids termed LY6D-shRNA-1, and LY6D-shRNA-2.

Cell transfection. We purchased pCMV6-XL5-LY6D and pCMV-XL5-vector (empty vector) (ORIGENE, Rockville, MD, USA). For transfection experiments, lipofectamine 3000 (Invitrogen, Waltham, MA, USA) was used to transfect LY6D or empty vector into Panc-1 cells. Transduction efficiency was analyzed by PCR and western blot.

Quantitative real-time reverse transcriptase–polymerase chain reaction. Total RNA was extracted from cultured cells using TRIzolR RNA Isolation Reagents (Thermo Fisher Scientific) as previously described (18). Complementary DNA (cDNA) was synthesized from 10 ng of total RNA using a High Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Polymerase chain reaction (PCR) was conducted using the Light CyclerTM 2.0 System (Roche Applied Science, Basel, Switzerland) with the Thunderbird® SYBR® quantitative PCR (qPCR) mix (Toyobo Life Science, Osaka, Japan). For each experiment, data were normalized to the expression of the control gene GAPDH. The following primers were used: LY6D: forward, 5′-CATTGCTGCTCCTTGCAG-3′ and reverse, 5′-ATGCTTGCTTGCAGTTGCTGGAG-3′; GAPDH: forward: 5′-AGCCACATCGCTCAGACAC-3′ and reverse, 5′-GCCCAATACG ACCAAATCC-3′.

Immunohistochemical staining. Immunohistochemical staining was performed as previously described (19). Slides were incubated with the anti-LY6D rabbit antibody (1:500 dilution, HPA024755; Sigma-Aldrich, St. Louis, MO, USA), overnight at 4°C. As a positive control for LY6D, we used human esophageal squamous tissue, based on the database of the Human Protein Atlas (https://www.proteinatlas.org). Groups that were slightly stained were considered positive, and those that were not stained at all were considered negative.

Cell proliferation assay. We utilized the Cell Counting Kit-8 (DOJINDO, Kumamoto, Japan) following the provided protocol to quantify the number of viable cells. After a 2-h incubation in the assay solution, we assessed the number of viable cells in each well (n=6) by measuring the absorbance at 450 nm (OD 450) with a BIO-RAD Model 680 XR microplate reader.

Scratch wound healing assay. Cells were plated at a density of 5×105 cells per well in 6-well plates (n=6) and cultured until they reached confluence under standard conditions. For the scratch assay, a 200 μl pipette tip was used to create three distinct wounds per well, and cells were then cultured in DMEM containing 1% FBS to inhibit proliferation. The plates were rinsed with fresh DMEM containing 1% FBS to remove any non-adherent cells before taking each set of photographs. Cell migration was assessed by measuring the average distance between the wound edges in nine randomly selected areas per well.

Invasion assay. The cell invasion assay was performed in a 24-well Corning Matrigel invasion chamber (Corning Inc., Corning, NY, USA) with 8-micron pores. To the top chamber, we added 200 μl of cell suspension (5×104 cells/ml) in serum-free media (n=6). Next, to the lower chamber, we added 500 μl of FBS-supplemented media to serve as a chemoattractant. After 72 h, the supernatant was discarded, the cells in the upper chamber were removed using a cotton swab, and the cells on the lower surface were fixed and stained with hematoxylin eosin. The number of invaded cells was counted under a microscope, assessing six high-power fields per group. Cells were counted using ImageJ software.

Sphere formation assay. The sphere formation assay was performed as previously described (14). In brief, sorted cells were seeded into 96-well ultralow attachment plates (Corning Inc.) at a density of 100 cells per well. These cells were cultured in tumorsphere medium (DMEM/F-12) supplemented with 20 ng/ml human platelet growth factor, 20 ng/ml epidermal growth factor, G418, and 1% antibiotic-antimycotic solution, at 37°C in a humidified environment containing 95% air and 5% CO2. We counted the number of spheres in each well and assessed the differences in the average sphere count per well (n=6).

Drug sensitivity assay. LP/CD44+ Panc-1, cells were seeded at a density of 2.0×103 cells/well in 96-well plates, and pre–cultured for 24 h (n=6, each). The cells were exposed to various concentrations of oxaliplatin and gemcitabine, and the cytotoxic effects were evaluated using a Cell Counting Kit-8 (DOJINDO, Japan), according to the manufacturer’s protocol.

Animal experiments. Animal experiments were performed in 8-week-old male BALB/cAJcl-nu/nu immunodeficient mice (CLEAJapan, Tokyo, Japan). To produce tumors in vivo, cells were mixed with Matrigel (BD Biosciences) and medium at a 1:1 ratio (vol:vol). Mice were subcutaneously injected with approximately 1.0×103 cells (sh group and empty vector control group, n=3 for each) in 100 μl medium/Matrigel solution, in both sides of the lower back regions. On week 6, the mice were sacrificed and the tumors were excised. Collected tissues were homogenized using a TissueLyser II (Qiagen Inc., Valencia, CA, USA).

Western blotting. A total of 60 μg of protein was extracted from cultured cells using RIPA buffer with added protease and phosphatase inhibitors (Thermo Fisher Scientific). The proteins were separated on 10% SDS-PAGE gels and transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany) at 100V for 90 min. β-actin was used as a loading control, and β-actin antibodies (A2066; Sigma-Aldrich) were employed. After blocking with 5% skim milk for 1 h, the membranes were incubated overnight at 4°C with primary antibodies at appropriate dilutions (1:1,000 for LY6D, 1:1,000 for CD24, and 1:2,000 for β-actin). Following incubation with secondary antibodies, protein bands were detected with the Amersham ECL Detection System (Amersham Biosciences, Piscataway, NJ, USA).

Prognostic analysis of LY6D. To examine LY6D expression in normal and tumor tissues, we used clinical information about tumors downloaded from the TCGA database (20). We analyzed the correlation between LY6D expression and PDCA prognosis, using overall survival (OS).

Gene mutation analysis of LY6D. We analyzed mutations of the LY6D gene in all tumor tissues using the cBioPortal platform (21, 22). The identified alterations included mutations, amplifications, deep deletions, and multiple alterations.

Statistical analysis. Statistical analyses were performed using JMP Pro 16.0.0 (SAS Institute Inc., Cary, NC, USA). We conducted an overall survival analysis, including all patients (n=75), using the Kaplan–Meier method. The log-rank test was used to test differences between the survival curves. Data are reported as mean±standard error of the mean (SEM). Variables that were significantly correlated with survival in univariate analysis were entered into a Cox proportional hazards regression model for multivariate analysis. A p-value of <0.05 was considered to indicate a statistically significant difference.

Approval of the research protocol by an Institutional Reviewer Board. The study was approved by the Institutional Review Board for Studies in Humans at Osaka University (approval number: 15144-6).

Informed consent. Informed consent was waived owing to the retrospective nature of the study. The opt-out recruitment method was applied to all patients, with an opportunity to decline to participate.

Animal studies. This study was approved by the institutional review board of our institution (Permission No. #15218).

Results

Study design. A flowchart of this study is shown in Figure 1.

Figure 1.
Figure 1.

Flowchart of the research.

LY6D is specifically up-regulated in tumor tissues. We employed TCGA to study LY6D expression in tumors and found that LY6D expression was up-regulated in almost all tumors compared to corresponding normal tissues (Figure 2A). We also analyzed the genetic alteration status of LY6D in different tumor samples from the cBioPortal platform. Amplification of LY6D constituted for the largest proportion among all mutation types, observed in various cancerous lesions, including pancreatic cancer (Figure 2B).

Figure 2.
Figure 2.

LY6D expression overview. (A) LY6D mRNA expression is significantly higher in human cancers than in normal tissues. CESC: Cervical squamous cell carcinoma and endocervical adenocarcinoma; LUSC: lung squamous cell carcinoma; ESCA: esophageal carcinoma; LUAD: lung adenocarcinoma; PAAD: pancreatic adenocarcinoma; UCEC: uterine corpus endometrial carcinoma; THCA: thyroid carcinoma; STES: stomach and esophageal carcinoma; READ: rectum adenocarcinoma; COADREAD: colon and rectum adenocarcinoma; COAD: colon adenocarcinoma; BLCA: bladder urothelial carcinoma; STAD: stomach adenocarcinoma; HNSC: head and neck squamous cell carcinoma; BRCA: breast invasive carcinoma; PRAD: prostate adenocarcinoma. (B) Alteration frequency of LY6D.

Overexpression of LY6D-enhanced the malignant potential of PDAC cells. To elucidate how LY6D is involved in cancer progression, we performed in vitro functional assays with manipulation of LY6D gene expression. We observed that LY6D overexpression at both the mRNA and protein levels significantly enhanced cell proliferation (Figure 3A, B). LY6D overexpression also enhanced the invasion and migration ability of Panc-1 cells (Figure 3C, D). Notably, LY6D expression in Panc-1 cells was significantly enhanced under 3D stem cell culture conditions compared to in 2D monolayer cultures (Figure 4A). Sphere formation assays revealed that LY6D overexpression significantly increased the number of spheroids (Figure 4B). Representative stem cell markers of PDAC including CD24, CD44, CD44v9, and CXCR4 were significantly enriched in LY6D overexpressing cells (Figure 4C).

Figure 3.
Figure 3.

Effect of LY6D overexpression on pancreatic ductal adenocarcinoma (PDAC) cells. (A) The efficacy of LY6D overexpression (OE) in Panc-1 cells, compared to in negative controls (NC), was determined by real-time PCR and western blotting (mean±SD; **p<0.01, two-tailed Student’s t-test). (B) Effect of LY6D OE on PANC-1 cells proliferation (mean±SD; *p<0.05, two-tailed Student’s t-test). (C) Representative images of the scratch wound healing assay, using LP/CD44+ cells transfected with NC and LY6D OE (Magnification: 100×). Average distance between wound edges in six different areas at the indicated time-points (relative change from the distance at 0 h) (mean±SD; *p<0.05, two-tailed Student’s t-test). (D). Representative image of NC and OE cells in Matrigel invasion assay for 72 h (mean±SD; *p<0.05, two-tailed Student’s t-test).

Figure 4.
Figure 4.

Overexpression of LY6D affects involvement in stemness. (A) Panc-1 cells in 3D culture had greater LY6D upregulation than Panc-1 cells in 2D culture (mean±SD; **p<0.01, two-tailed Student’s t-test). (B) Overexpression (OE) cells had a stronger sphere-forming ability than negative control (NC) cells. The y-axis shows the average number of spheres per well. (C) Cancer stem cell (CSC) marker analysis showed that OE cells had upregulated CSC markers (mean±SD; *p<0.05, **p<0.01, two-tailed Student’s t-test).

Stem cell properties of LP/CD44+ cells. Using the ODC-degron system, we previously identified that cells exhibiting low proteasome activity possess high stem cell properties (14, 17). Here we used this ODC-degron system to conduct a more detailed investigation into the association of LY6D with stemness. These cells were also gated using flow cytometry with the CD44v9 antibody, a representative PDAC stem cell marker. (Figure S1A, B).

The sphere formation ability was significantly increased in LP/CD44+ cells compared with parent cells (Figure S2A). We also further examined the expressions of various cancer stem cell (CSC) markers in LP/CD44+ cells and parent cells. Representative stem cell surface markers in PDAC (CD24, CD133, and CXCR4) were significantly enriched in LP/CD44+ cells compared with parent cells (Figure S2B). Sphere formation assay and CD markers results indicated the stemness of LP/CD44+ cells. Furthermore, compared to parent cells, LP/CD44+ cells showed enhanced drug resistance to the anticancer drugs oxaliplatin and gemcitabine (Figure S2C). We additionally analyzed the potential effect of LY6D on Panc-1 cell migration by performing a wound-healing assay, which demonstrated that LP/CD44+ cells displayed increased migration ability (Figure S3A). We confirmed a significant elevation of LY6D expression in LP/CD44+ cells (Figure 5A). Furthermore, LY6D expression was significantly enhanced under 3D stem cell culture conditions compared to in 2D monolayer cultures in LP/CD44+ cells (Figure S3B).

The effects of LY6D knockdown on cancer stem cells. LY6D knockdown experiments were performed in LP/CD44+ cells, which showed high LY6D expression levels. RNA interference (RNAi)-mediated gene silencing suppressed the gene expression by 50% in sh1 cells and by 70 % in sh2 cells (Figure 5B). Sphere formation assays revealed that LY6D knockdown significantly decreased the number of spheroids formed (Figure 5C). LY6D knockdown also inhibited the invasion and migration ability of LP/CD44+ cells (Figure 6A, B). Furthermore, LY6D knockdown suppressed the cells’ resistance to the anticancer drug gemcitabine to lower than in NC cells (Figure 6C). To investigate whether LY6D played a role in tumor formation in vivo, we subcutaneously injected LP/CD44+ cells with stable LY6D knockdown or NC cells into the bilateral hind legs of nude mice (n=3). LY6D knockdown significantly decreased the volume of PDAC tumors in this xenograft mouse model (Figure 6D).

Figure 5.
Figure 5.

Knockdown of LY6D affects sphere formation. (A) LY6D expression in LP/CD44+ cells was determined by real-time PCR and western blotting (mean±SD; **p<0.01, two-tailed Student’s t-test). (B) LY6D knockdown in LP/CD44+ cells was detected by real-time PCR and western blotting. (C) LP/CD44+ cells had stronger sphere-forming ability than shNC cells. The y-axis shows the average number of spheres per well (mean±SD; *p<0.05, two-tailed Student’s t-test).

Figure 6.
Figure 6.

Knockdown of LY6D affects pancreatic ductal adenocarcinoma (PDAC) cells. (A) Representative images of the scratch wound-healing assay using LP/CD44+ cells transfected with negative control (NC) and LY6D shRNA (Magnification: 100×). Average distance between wound edges determined for six different areas at the indicated time points (relative change from the distance at 0 h) (mean±SD; *p<0.05, **p<0.01, two-tailed Student’s t-test). (B) Representative image of cells in Matrigel invasion assay of NC and LY6D shRNA cells for 72h.(C) Dose response curve after exposure to gemcitabine. (D) In vivo analysis using a xenograft model mice (n=3). Tumor size in NC and LY6D shRNA cells (mean±SD; *p<0.05, two-tailed Student’s t-test).

Immunohistochemical analysis for LY6D expression in PDAC. LY6D expression was not detected in normal tissues (Figure 7A). In contrast, 26 of 75 PDACs (34.6%) exhibited positive staining for the LY6D protein, and the majority of tumors exhibited heterogeneous LY6D expression (Figure 7A). Moreover, LY6D expression was positively correlated with the expression of CD24 (Figure 7B).

High LY6D expression was associated with poor prognosis in PDCA from the TCGA datasets (Figure 7C). We also showed that positive LY6D expression was significantly associated with lower overall survival rate (p=0.03) and lower relapse-free survival rate (p=0.005) (Figure 7D, E). Univariate analyses indicated that LY6D expression was a significant prognostic factor for overall survival (p=0.0092) and relapse-free survival (p=0.0156) (Table I). In multivariate analysis, LY6D expression remained an independent prognostic factor for overall survival (p=0.0012) and relapse-free survival (p=0.0025) (Table I). Clinicopathological analyses indicated no significant association between LY6D expression and various clinicopathological features of PDAC (Table II).

Figure 7.
Figure 7.

Predominant expression of LY6D in cancer tissues and its clinical significance. (A) Representative images of immunohistochemical staining of LY6D, showing negative results in normal tissues and heterogenic expression in cancer tissues (Magnification ×4; scale bar indicates 500 μm). PDAC, Pancreatic ductal adenocarcinoma. (B) LY6D and CD24 markers are co-expressed in PDAC. (C) Kaplan–Meier curves for overall survival of PDAC patients according to TCGA of LY6D. (D) Kaplan–Meier curves for overall survival of PDAC patients (n=75) according to immunohistochemical staining of LY6D. (E) Kaplan–Meier relapse-free survival curves of PDAC patients (n=75).

View this table:
Table I.

Survival analyses. Univariate and multivariate analyses for overall and relapse-free survival (Cox regression model).

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Table II.

Relationship between Ly6D expression and clinicopathological features in pancreatic ductal adenocarcinoma.

Discussion

In addition to conventional therapy, there is growing demand for novel targeted therapies for pancreatic cancer (23). Despite increasing evidence in this field, the currently available results are not satisfying in terms of evaluating therapeutic strategies to target CSCs (24, 25). CSCs are responsible for sustaining the tumorigenicity of the tumor and generating the diverse cell types that comprise the tumor (7). Here we established a population of cancer cells with extremely high tumorigenic potential, with specific focus on immune-related LY6D gene. Our findings revealed that LY6D plays a crucial role in maintaining the stem cell properties of these cells.

LY6 family genes were initially identified in mice as lymphocyte differentiation antigens and as membrane-bound proteins with GPI-anchors (26, 27). The original member of the LY6 protein family, CD59, was discovered in human lymphoid cells, where it plays roles in the complement membrane attack complex and T cell activation (28, 29). Currently, 20 human LY6 proteins have been identified, with molecular weights ranging from 11-36 kDa, and they have been categorized as either transmembrane or secretory based on the presence of a GPI-anchored signal sequence (30, 31). LY6 family genes are located on chromosome 8q24 alongside c-Myc. Somatic copy number gain in the 8q region is the most common type of copy number gain in multiple types of cancer (10). LY6D is located on 8q24.3, a region that is frequently amplified in various types of human malignancies (32).

The precise function of LY6D in carcinogenesis remains largely unknown. Cell invasion and migration are crucial processes for cancer cell metastasis (33). Our vitro studies showed that LY6D expression was closely associated with drug resistance, migration, and invasion ability of pancreatic cancer cells. Furthermore, LY6D knockdown inhibited pancreatic stem-like cancer cell treatment resistance, migration, and invasion. On the other hand, LY6D overexpression promoted invasion and migration ability. These findings suggested that LY6D is involved in PDAC cell progression and invasion. Previous studies have demonstrated that elevated LY6D expression is significantly correlated with increased risk of tumor recurrence, and poor prognosis in breast and lung cancers (11, 12, 31, 34). In our present study, we showed that LY6D positivity group was related to poor prognosis and high relapse rate in PDAC. Multivariate analysis indicated that LY6D was an independent prognostic factor for overall survival. Our present findings suggest that abnormal LY6D expression may represent a promising new predictive biomarker and therapeutic target for pancreatic cancer.

Overall, our findings showed that LY6D is highly expressed in pancreatic cancer stem cells and is associated with tumor malignancy. Further research is needed to reveal the detailed regulatory mechanisms of LY6D, and to explore the potential development of cancer drugs targeting LY6D.

Acknowledgements

This study was partly supported by a research grant from Kagoshima Shinsangyo Sousei Investment Limited Partnership (its general partner is Kagoshima Development Co., Ltd).

Footnotes

  • Authors’ Contributions

    SO, NN, and HY provided conception and design of the study, and contributed to acquisition and analysis of data. HT, AH, TO, NM, MU, and MM contributed to acquisition and analysis of data. SO, NN and HY wrote the article. All authors were engaged in interpretation of data, revising the article, approved the final version of article, and agreed in all aspects of the work. HY and YY performed ODC degron-related experiments. HT, SK, YD and HE collected and provided clinical samples and data.

  • Supplementary Material

    Available at: https://figshare.com/s/69491fa2a61edaa9e9f5

  • Conflicts of Interest

    The Authors declare no conflicts of interest associated with this manuscript.

  • Received July 15, 2024.
  • Revision received August 21, 2024.
  • Accepted September 11, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Lymphocyte Antigen 6 Family Member D (LY6D) Affects Stem Cell Phenotype and Progression of Pancreatic Adenocarcinoma
SHUMPEI OKIMURA, NAOHIRO NISHIDA, HIDEKAZU TAKAHASHI, YUHKI YOKOYAMA, HIROYUKI YAMAMOTO, ATSUSHI HAMABE, TAKAYUKI OGINO, NORIKATSU MIYOSHI, HIDENORI TAKAHASHI, MAMORU UEMURA, SHOGO KOBAYASHI, MASAKI MORI, YUICHIRO DOKI, HIDETOSHI EGUCHI, HIROFUMI YAMAMOTO
Anticancer Research Nov 2024, 44 (11) 4737-4749; DOI: 10.21873/anticanres.17300
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