CPI-613 Suppresses Pancreatic Cancer Stem-Like Cells by Targeting the SEMA3A/NRP1 Pathway

doi:10.21203/rs.3.rs-3804185/v1

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CPI-613 Suppresses Pancreatic Cancer Stem-Like Cells by Targeting the SEMA3A/NRP1 Pathway

https://doi.org/10.21203/rs.3.rs-3804185/v1

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Pancreatic cancer is known for its recurrence and chemotherapy resistance, partly attributed to cancer stem cells (CSCs). Targeting CSCs could improve the efficacy of standard cytotoxic therapies and potentially prevent or delay cancer recurrence. The increased reliance on the modulation of metabolic pathways in CSCs offers a potential therapeutic opportunity. In this study, we investigated the effects of CPI-613 (Dedvimistat), a metabolic inhibitor that targets the tricarboxylic acid cycle on pancreatic cancer cells. We demonstrate that treatment with CPI-613 significantly reduced the capacity to form CSCs, furthermore, CPI-613 showed decreased tumorigenicity in vivo. To gain further insights into the molecular mechanisms underlying the effects of CPI-613, RNA-Seq analysis was performed. CPI-613 impeded the unintended enrichment of CSC through target SEMA3A (Semaphorin 3A) and its receptor NRP1 (Neuropilin 1). Knockdown of SEMA3A significantly inhibited the proliferation of CD44 + frequency and increased their sensitivity to gemcitabine. Moreover, treatment with exogenous SEMA3A counteracted the inhibitory effect of CPI-613 on CSC proliferation and promoted resistance to gemcitabine. Our findings suggest that CPI-613 selectively targets pancreatic CSCs through the SEMA3A/NRP1 pathway. CPI-613 has the potential to overcome chemoresistance and prevent cancer recurrence by targeting CSCs.

Pancreatic cancer remains one of the deadliest cancers with a significantly poor prognosis^[1]. Current primary treatment options include surgical removal and chemotherapy. Unfortunately, the lack of clear symptoms and reliable early detection techniques frequently results in late-stage diagnoses, rendering the disease inoperable, which contributes to a poor survival rate^[2]. Furthermore, numerous studies suggest the presence of a unique subset of cells within pancreatic tumors, possessing the ability for self-renewal, differentiation, and driving tumor initiation and metastasis^[3]. These stem cell-like tumor cells are believed to be a primary factor in resistance to chemotherapy and radiation therapy^[4–6]. We hypothesize that targeted suppression of these cancer stem cells (CSCs) represents a promising therapeutic strategy, with the potential to prevent the recurrence of pancreatic cancer. Furthermore, a comprehensive elucidation of the molecular characteristics associated with the CSC phenotype is vital to developing effective treatments.

According to the Seed and Soil concept, cancer cells within a primary tumor transform and develop a unique metabolic signature that paves the way for their future settlement in a distinct organ^[7]. Only those cancer cells with metabolic adaptability can successfully inhabit distant organs and generate metastases. The structure and composition of the CSCs niche can vary significantly depending on the tissue type, adapting to the distinct functions and requirements of the specific tumor^[8]. CSC niche is believed to be influenced by both intrinsic and extrinsic mechanisms. Intrinsic factors involve pluripotency factors like Sox2, c-MYC, and KLF4^[9]. The expression of these transcription factors can reprogram cells into CSCs and promote cellular plasticity, enabling tumor cells to adapt and survive in challenging environments. Indeed, elevated levels of Sox2, Nanog, and OCT4 have been demonstrated to promote stemness development and induce resistance to drugs in various solid tumors^[10].

CPI-613, also known as Devimistat, represents the inaugural member of lipoic acid derivatives that exert potent inhibitory effects on tumor growth by modulating mitochondrial enzyme activity and the redox state^[11]. CPI-613 operates as a targeted inhibitor of the mitochondrial tricarboxylic acid (TCA) cycle, a critical process in cancer metabolism, by specifically targeting the enzymes pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (α-KGDH)^[12]. Extensive preclinical research has provided compelling evidence of CPI-613's remarkable ability to inhibit tumor growth while exhibiting minimal side-effect toxicity. Notably, in a Phase I trial, patients with metastatic pancreatic cancer treated with CPI-613 and modified FOLFRINOX achieved an impressive objective response rate of 61%, which included a notable 17% complete response rate^{[13, 14]}. However, the precise molecular mechanisms underlying the action of CPI-613 are yet fully understood.

In this study, we aim to investigate CPI-613 selectively targeting pancreatic cancer stem cells and enhance the efficacy of chemotherapy. Our finding provides compelling evidence that CPI-613 indeed preferentially targets pancreatic CSCs. In addition, our observations indicate that CPI-613 exerts an inhibitory effect on the proliferation of CSCs by modulating the SEMA3A/NRP signaling pathway. Our finding highlights the potential of combining CPI-613 with chemotherapy as a promising therapeutic strategy for the treatment of pancreatic cancer.

2.1 Cell Lines and Sphere Formation Assays

Human pancreatic cancer cell lines BxPC-3 and PANC-1 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured at 37°C in 5% CO₂ Dulbecco modified Eagle medium (Gibco, Carlsbad, CA) with 10% fetal bovine serum (FBS). For sphere formation, single-cell suspensions were cultured in serum-free DMEM coated with 0.7% agar plates at a density of 7×103/100 mm, Spheres were analyzed after 5 days.

2.2 Colony Formation Assay

5000 cells mixed with agar were seeded onto six-well plates (Coring) and cultured for 2 weeks, and fed with 1 ml of the assay medium twice a week. The presented colonies were fixed with 4% paraformaldehyde for 20 minutes and stained with crystal violet.

2.3 Drug Treatment

CPI-613 was purchased from Selleckchem, and IC50 values were determined by Cell. All the cell lines were plated in 96-well plates at a density of 1×104 cells/well and treated with CPI-613 for 24 h, 48 h, and 72 h, cell viability was determined by ATP by CellTiter-Glo Luminescent Cell Viability Assay (Promega).

2.4 RNA isolation and quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from samples using Trizol reagents (Invitrogen) according to the manufacturer’s instructions. One microgram of RNA was then reverse-transcribed to cDNA by ReverTra Ace qPCR RT Master Mix (TOYOBO). Real quantitation of all transcripts was detected via real-time RT-PCR performed using FastStart Universal SYBR Green Master (Roche) kit on the ABI quantitative PCR system. Relative mRNA transcript levels were normalized to GAPDH expression by means of the 2-ΔΔCt method. PCR primer sequences are listed in Additional File 1: Table S1.

2.5 Western Blotting

Proteins were extracted with lysis buffer (50 mM Tris Ph 7.4) supplement with phosphatase inhibitor cocktail and quantified using a BCA protein assay kit. 30 µg protein was separated on SDS-PAGE with 10% stacking gel and membranes were blocked in 5% skimmed milk powder. Then the membrane was incubated overnight probing at 4°C with primary antibodies, followed by incubation with either a mouse or rabbit horseradish peroxidase (HRP)-conjugated secondary antibody and detection with ECL Prime (Thermo). The following primary antibodies were used: CD24, CD44, ESA, OCT4, SEMA4A, and Actin. Protein bands were quantified by ImageJ software.

2.6 Tumorigenicity in Vivo Assay

Five-week-old BALB/c nude mice were randomly grouped into 2 arms. Cells were injected subcutaneously into the flank. The tumor volume was calculated using the following formula: (width2 × height)/2. For treatment with CPI-613, when the tumor volume reached 100–150 mm3, CPI-613 was administered at 25 mg/kg two times per week, for two weeks of drug treatment, the mice were euthanized and tumors were harvested for further analyses.

2.7 Immunohistochemistry analysis

Each xenograft was fixed with paraffin-embedded and cut into 5 µm sections. The sections were deparaffinized and re-hydrated, and the antigen was retrieved with citrate buffer. After blocking with serum for 1 h, the sections were placed into a wet box and incubated overnight with CD44 primary antibodies, the primary antibodies were washed away and added with biotinylated secondary antibodies for 1 h. Finally, the sections were stained with DAB and hematoxylin. the staining intensity was scored as follows: 0 for none, 1 for weak brown, 2 for moderated brown, and 3 for strong brown. All images were obtained with a photographic microscope and analyzed using Image J.

2.8 Statistics analysis

All the results were analyzed with GraphPad Prism 7.0 (GraphPad Software). Data obtained from the experiments were analyzed using Student’s t-tests and one-way ANOVA. A difference of P < 0.05 was considered statistically significant.

3.1 CPI-613 induces a decrease in PDAC stem-like positive cell frequency

As demonstrated in the previous studies^[15], CPI-613 inhibited cell proliferation of pancreatic cell lines in a dose and time-dependent. Our results showed the growth of adherent BxPC-3 cells can be inhibited at the concentration of 70 µM and 90 µM CPI-613. Notably, we observed no significant inhibition at a concentration of 60 µM.

Since sphere formation is considered a selection method that enriches CSCs^[16], we performed sphere-forming cultures using BxPC-3 cells to investigate the stem-like properties of PCSCs. We measured the spheroid formation ability in the presence and absence of CPI-613, compared with the control group, the spheroid formed in the 30 µM and 60 µM treatment groups were smaller (Fig. 1A and 1B). It was also found that 60 µM significantly inhibited colony formation (Fig. 1C, P > 0.001). Therefore, we hypothesized that CPI-613 would preferentially target stem-like cancer cells. To test this hypothesis, the cells were harvested for CPI-613 after 48 hours. Treatment with CPI-613 induced a decrease in cancer stem cell markers including CD44, ESA, OCT4, and SOX2 (Fig. 1E, 1F, and 1G). These data confirmed that CPI-613 targets pancreatic cancer stem-like cells in our model.

To investigate the impact of CPI-613 on the tumorigenicity of PCSCs, we conducted an in vivo study involving the subcutaneous injection of BxPC-3 cells into BALB/c nude mice (Fig. 2A). Upon reaching a tumor volume of 200 mm³, the mice received CPI-613 treatment at a dose of 12.5 mg/kg administered every three days. Our findings revealed that tumor growth was notably more rapid in control groups than compared to the CPI-613 treatment groups (Fig. 2B). Subsequently, we conducted a comprehensive assessment of tumor volume and tumor weight between the two groups (Fig. 2C-2D), which unequivocally demonstrated that CPI-613 exerted a significant inhibitory effect on the proliferation of pancreatic cancer cells. This observation was further corroborated through histopathological examination using H&E staining. Additionally, we observed a marked reduction in the expression of CD44 in response to CPI-613 treatment (Fig. 2E). These findings are consistent with our in vitro analysis, confirming the ability of CPI-613 to reduce the frequency of CSCs. Taken together, our findings suggest that CPI-613 holds substantial potential as a therapeutic agent for pancreatic cancer by selectively targeting CSCs.

3.2 CPI-613 treatment induced a decreased expression of SEMA3A in CSCs

To gain further insights into the underlying mechanism of CPI-613 in pancreatic CSCs, we conducted RNA sequencing (RNA-Seq) analysis. By comparing the gene expression profiles of cells treated with CPI-613 to those of control cells, we identified 8 genes that exhibited significant downregulation in the CPI-613 treatment group. These genes were SEMA3A, MAP2K6, TXNIP, AQP3, NTRK1, EFMP1, DOCK4 and SOX2 (Fig. 3A). Among these genes, we focused our attention on SEMA3A due to its frequent expression across various cancers and its known association with cancer aggressiveness, primarily through its role in promoting angiogenesis and cell migration^{[17, 18]}. Furthermore, a recent study has revealed that SEMA3A stimulates the division and expansion of CSCs^[19]. Our RNA sequencing data substantiated these findings (Fig. 3B), and gene set enrichment analysis (GSEA) provided additional support for the involvement of SEMA3A in the regulation of cell cycle processes and RHO-GTPASE signaling (Fig. 3C). Collectively, these results suggest that CPI-613 may exert its effects on pancreatic CSCs by modulating the expression of SEMA3A, thereby influencing the crucial role of cellular functions.

3.3 Prognostic values of SEMA3A expression in patients with pancreatic cancer

We conducted a study to investigate the role of SEMA3A in the context of pancreatic cancer. SEMA3A is a member of the Semaphorin proteins family, characterized by the presence of a 500 amino acid sema domain^[20]. Its cognate holoreceptor complex is comprised of Neuropilin-1 (Nrp1) and PlexinA1(PlxnA1)^{[21, 22]}. Notably, SEMA3A is known to bind exclusively to Nrp1. Our investigation evaluated the expression and prognostic significance of SEMA3A and Nrp1 in pancreatic cancer.

We collected information on 183 patients with pancreatic cancer from the TCGA database (Normal = 4, Tumor = 179). Our analysis revealed that SEMA3A displayed significantly higher expression levels in pancreatic cancer tissues in compared to normal tissues (Fig. 4A). Moreover, elevated SEMA3A expression correlated with poor overall survival (OS), disease-specific survival (DSS), and progress-free interval (PFI) in pancreatic cancer patients (Fig. 4B). To assess the diagnostic potential of SEMA3A, we constructed a receiver operating characteristic (ROC) curve, which effectively differentiated SEMA3A expression in normal and pancreatic samples. The area under the curve (AUC) value for SEMA3A levels was notably high at 0.973 (CI: 0.955–0.992), indicating its strong diagnostic potential (Fig. 4C).

Additionally, our investigation extended to the examination of SEMA3A protein expression, which was found to be upregulated in pancreatic cancer tissue compared to normal tissues (Fig. 4D). Furthermore, we explored the co-expression of SEMA3A and NRP1 in PDDA. Our analysis unveiled a positive correlation between SEMA3A and NRP1 expression (r = 0.532) (Fig. 4E). Consistent with this, NRP1 mRNA expression exhibited a significant upregulated in pancreatic tissues compared to normal tissues (Fig. 4F). Increased NRP1 expression was associated with poorer disease-free survival (Fig. 4G), and the protein expression levels of NRP1 were also elevated in pancreatic tissues (Fig. 4H). To further elucidate the role of NRP1 in pancreatic cancer across different clinical characteristics, we utilized the UALCAN database. Our analysis revealed that significantly differential expression of NRP1 in pancreatic cancer samples compared to normal samples, as well as variations in different stages and grade statuses of pancreatic cancer (Fig. 4I).

3.4 SEMA3A is critical for the maintenance of pancreatic CSC properties

In our current investigation, we examined the expression profiles of SEMA3A and NRP1 in both normal and pancreatic cancer cell lines. Our results revealed a notable upregulation of both mRNA and protein levels (Fig. 5A-5C) in pancreatic cancer cell lines as compared to normal cells. To further elucidate the potential effects of CPI-613 on pancreatic cancer stem cells, we explored the expression of SEMA3A and NRP1. Intriguingly, our findings demonstrated a significant reduction in mRNA expression within cells treated with CPI-613, as opposed to those treated with DMSO control (Figs. 5D and 5E). This reduction in mRNA expression was consistently reflected in decreased protein levels of SEMA3A and NRP1 in CPI-613 treated cells (Fig. 5F and 5G). Collectively, our findings provide robust evidence that CPI-613 exerts inhibitory effects on the proliferation of pancreatic cancer stem cells by suppressing the expression of SEMA3A and NRP1. Further studies are warranted to fully elucidate the precise mechanisms of SEMA3A and NRP1 as targets of CPI-613.

3.5 CPI-613 inhibited pancreatic CSCs by decreased SEMA3A/NRP1 expression

To investigate the role of the SEMA3A/NRP1 axis in governing the proliferation of pancreatic CSCs and its interaction with CPI-613, we transfected the CSCs with SEMA3A siRNA or empty vector to assess the effectiveness of SEMA3A knockdown (Fig. 6A). Notably, the knockdown of SEM3A resulted in a decrease in CSCs. To further examine the effect of SEMA3A suppression on CSC characteristics, we explored its influence on sphere formation, a hallmark of CSCs (Fig. 6B). Additionally, we evaluated the effect of SEMA3A on the expression of key stemness factors, including OCT4, and SOX2, as well as the surface marker CD44 and ESA. Utilizing qRT-PCR and western blot analysis, we discerned that the downregulation of SEMA3A expression effectively inhibited the enrichment of CSC markers in BxPC-3 and Capan-2 cells (Figs. 6C and 6D). Notably, the knockdown of SEMA3A also resulted in a decreased population of CD44 + cells in both BxPC-3 and Capan-2 cells (Fig. 6E). Furthermore, our investigation extended to examining the influence of SEMA3A on chemoresistance. Remarkably, we observed a significant increase in Caspase-3 expression in siRNA-treated cells, indicating enhanced sensitivity to gemcitabine-induced apoptosis. Taken together, these findings suggest that the SEMA3A/NRP1 axis plays a crucial role in regulating pancreatic sphere formation and chemoresistance.

3.6 SEMA3A recuse the inhibition of CPI-613 on pancreatic CSCs proliferation

Given that SEMA3A is a potential target for CPI-613, we treated BxPC-3 and Capan-2 cells with recombinant SEME3A proteins to assess their impact on CSCs properties in the presence of CPI-613. The results revealed that SEMA3A treatment rescued the inhibitory effect of CPI-613 on pancreatic sphere formation (Fig. 7A). To corroborate these findings, we examined the expression levels of SEMA3A and its receptor NRP1 at both mRNA and protein levels upon exposure to SEMA3A recombinant proteins (Fig. 7B and 7C). Significantly, we observed an upregulation of SEMA3A and NRP1, further validating the enhanced expression of these key components upon SEMA3A supplementation. Additionally, our investigation extended to evaluating the mRNA and protein levels of CSC markers, including CD44, ESA, SOX2, and Oct4. Remarkably, SEMA3A treatment led to their upregulation in CPI-613-treated cells. This suggests that SEMA3A supplementation enhances the expression of the CSCs markers (Figs. 7B and 7C). Moreover, flow cytometric analysis revealed that the SEMA3A group exhibited a significant increase in the CD44 + cell population when treated with CPI-613, further supporting the role of SEMA3A in promoting CSC properties (Fig. 7D). SEMA3A treatment was also found to decrease the expression level of Caspase-3 when cells were exposed to gemcitabine, suggesting that SMEA3A may confer resistance to gemcitabine-induced apoptosis in CPI-613 treated cells (Fig. 7E). Collectively, these findings provide further evidence that CPI-613 suppresses pancreatic CSCs and enhances the therapeutic efficacy of gemcitabine through the SEMA3A/NRP1 pathway. These results not only deepen our understanding of the mechanisms underlying the therapeutic potential of CPI-613 in targeting pancreatic cancer but also offer valuable insights for the development of novel therapeutic strategies in pancreatic cancer treatment.

Gemcitabine, a chemotherapy agent, has been widely used as the standard treatment for advanced pancreatic cancer. However, its effectiveness is significantly hampered by the development of resistance mechanisms that are not fully elucidated. Emerging evidence suggests that cancer stem cells (CSCs) may play a pivotal role in the acquisition of gemcitabine resistance. These CSCs exhibit distinct phenotypic characteristics that enable them to evade conventional treatment approaches. Recent investigations have shed light on the unique energy metabolism features of CSC populations. In normal cells, oxidative phosphorylation is the predominant pathway for ATP production, as it generates a higher yield of ATP compared to glycolysis. However, in the context of cancer, particularly in hypoxic microenvironments, there is a pronounced shift toward glycolysis as the primary energy source. This metabolic preference for glycolysis, even in the presence of oxygen, is a phenomenon known as the Warburg effect. This metabolic reprogramming towards glycolysis in cancer cells, including CSCs, contributes to their sustained survival and proliferation. Consequently, understanding these intricate metabolic alterations in CSCs may hold the key to devising more effective strategies for overcoming gemcitabine resistance in advanced pancreatic cancer.

Gemcitabine has long served as the established standard chemotherapy for the treatment of advanced pancreatic cancer. Nevertheless, the emergence of chemoresistance to gemcitabine poses a significant challenge, greatly diminishing its therapeutic efficacy. The precise mechanisms underlying the development of gemcitabine resistance remain a subject of ongoing investigation. It is widely acknowledged that an initially beneficial response to gemcitabine treatment may inadvertently promote the expansion of more aggressive clonal cell populations. This expansion is, in part, attributed to cancer stem cells (CSCs), which exhibit diverse resistant phenotypes and possess an inherent capacity to evade the detrimental effects of contemporary treatment strategies. Furthermore, CSCs serve as the reservoir for repopulating the tumor.

Recent research findings have shed light on the distinctive energy metabolism profiles observed within CSC populations. Under normal physiological conditions, cells obtain energy through the catabolism of nutrients, primarily via glycolysis and oxidative phosphorylation. In contrast to normal cells, which predominantly rely on oxidative phosphorylation due to superior ATP production yield, cancer cells, including CSCs, often proliferate within hypoxic environments and exhibit a heightened dependency on glycolysis. Remarkably, this metabolic preference for glycolysis persists even in the presence of oxygen, a phenomenon known as the Warburg effect. The adoption of anaerobic metabolism, albeit less efficient in terms of ATP generation compared to oxidative phosphorylation, appears to confer advantages to rapidly dividing cells and their metastatic potential by generating elevated levels of lactate, thus acidifying the tumor microenvironment.

Notably, CSCs appear to favor mitochondrial oxidative metabolism as their primary energy source. Moreover, when oxidative phosphorylation is inhibited, CSCs exhibit the capacity to adapt and switch to glycolysis, underscoring their remarkable metabolic plasticity. These insights into CSC metabolism not only enhance our understanding of the resistance mechanisms but also hold the potential to inform the development of novel therapeutic strategies aimed at targeting and overcoming gemcitabine resistance in advanced pancreatic cancer.

CPI-613, also recognized as delimitate, is a derivative of lipase known for its ability to inhibit crucial mitochondrial enzymes, namely pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, which play pivotal roles in mitochondrial metabolism. Our working hypothesis posits that pancreatic cancer stem cells (CSCs) inherently rely more heavily on mitochondrial oxidative phosphorylation, or can readily adapt to this metabolic pathway under stress conditions. Consequently, disrupting this pathway holds the potential to deplete cellular energy resources, thereby promoting cell death or rendering these cells more susceptible to apoptotic mechanisms.

Recent investigations have unveiled intriguing findings related to the impact of CPI-613 treatment. Notably, it has been observed that treatment with CPI-613 leads to a reduction in the frequency of CD133 + and CD117 + cells, notably affecting the ovarian CSC population. In our research endeavors, we subjected pancreatic cells to CPI-613 treatment, resulting in a marked decrease in the presence and enrichment of CSCs. Moreover, CPI-613 treatment led to a notable reduction in the expression levels of key CSC markers, including CD44, ESA, OCT4, and Sox2. Particularly noteworthy was the significant decrease observed in the CD44 + fraction of CSCs.Taken together, these compelling data reinforce the promising potential of CPI-613 as a strategic therapeutic approach for combatting pancreatic cancer. These findings not only underscore the importance of targeting mitochondrial metabolism in CSCs but also highlight CPI-613 as a promising candidate for further exploration and development as a therapeutic intervention in the battle against pancreatic cancer.

CD44, identified as a surface marker, plays a crucial role in the context of CSCs and emerges as a promising therapeutic target for the modulation of CSC ^[23]. Notably, in pancreatic cancer, high expression of CD44 is strongly associated with invasiveness, metastasis, and mesenchymal-like stem cell properties^[24]. CD44 plays multifaceted roles in cancer progression, metastasis, and resistance to conventional treatments, with its expression being upregulated in both human pancreatic cancer cell lines and tumor tissues^{[25, 26]}. Encouragingly, research studies have demonstrated significant therapeutic benefits by blocking CD44 using anti-CD44 monoclonal antibodies in the context of pancreatic cancer. In vitro data have unveiled compelling evidence that blocking CD44 leads to a substantial reduction in the growth and invasive capacity of pancreatic cancer cells. These findings highlight the promising potential of CD44 as a therapeutic strategy for pancreatic cancer. In our data, the administration of CPI-613 has been shown to effectively suppress CD44 expression in human pancreatic cancer cells. Worth noting is the correlation observed between CD44 expression levels and histologic grade, with patients harboring CD44-positive tumors displaying poorer prognosis outcomes^[27]. Thus, targeting CD44 represents a viable approach to counteract drug resistance in the treatment of pancreatic cancer, offering a promising avenue for improving patient outcomes.

Sema3a exhibits a dual role in tumorigenesis, acting as either a tumor-inhibiting or tumor-promoting factor contingent on the specific tumor microenvironment. Prior works had indicated that sema3 produced from the CSC niche, triggers symmetric divisions that facilitate the expansion of the CSC population. Stimulation with SEMA3A has been demonstrated to induce sphere formation in breast cancer cells through the NRP1 receptor. Notably, breast cancer patients displaying high NRP1 staining in their tumor tissues have exhibited unfavorable prognoses marked by relapse of drug resistance^[28]. In our current study, we provide evidence demonstrating that the downregulation of the SMEA3A receptor is sufficient to impede the proliferation of pancreatic CSCs. Furthermore, the results show that the treatment of pancreatic cancer cells with SEMA3A promotes CSC proliferation, an effect that is counteracted by CPI-613. These findings align with previous conclusions drawn from research on brain tumor stem cells, which express SEMA3A, its receptor NRP1, and PlxnA1. Notably, NRP1 is found in the CD133-positive brain cancer stem cell but absent in differentiated tumor cells. The loss of SEMA3A and its receptor has been shown to inhibit tumor growth and pro-invasive effects of brain cancer stem cells ^{[29, 30]}. Additionally, autocrine SEMA3A has been identified as a critical factor for sustaining the self-renewal capacity of Lewis lung carcinoma by modulating mTORC1 activity. However, other works have also highlighted SEMA3A as a tumor suppressor in the context of tumor angiogenesis^[31]. The dual functionality of SEMA3A appears to be contingent on the tumor environment and the specific receptor interactions involved.

In our preliminary studies, we present evidence that pancreatic cancer tissue exhibiting elevated levels of SEMA3A and NRP1 are associated with poor prognosis and clinical characteristics. SEMA3A/NRP1 signaling may be actively involved in a subset of pancreatic CSCs. Our results reveal that pancreatic CSCs can be effectively targeted using CPI-613, which acts as modulating their distinct mitochondrial metabolism through the regulation of SEMA3A/NRP1 activity. These results hold promise for reducing the bulk tumor cell population while concurrently targeting CSCs, which have the potential to replenish daughter cells and prevent tumor recurrence, ultimately leading to more enduring treatment responses.

Funding

This work was supported by funds from the Guangdong Basic and Applied Basic Research Foundation, China（No. 2021B1515140031 and 2020A1515110029）, Dongguan Science and Technology of Social Development Program, China (No. 2019507163147 and 20211800907773).

Authors contribution

Zhuanghua Li: Conceptualization, Data curation. June Wang: Writing - Original draft preparation. Baocheng Xie: Methodology, supervision. Hongyu Liu: Methodology and investigation, formal analysis. Zhuo Dong: Writing – review & editing. Linxuan Huang: Conceptualization, Project administration, Funding acquisition, Writing- review & editing.

Availability of data and materials

Not applicable

Ethics approval and consent to participate

Thiswork was approved by the The Tenth Affiliated Hospital of Southern Medical University (ethical approval number: IACUC-AWEC-202305006)

Consent for publication

All the authors have read and approved the submission of the current manuscript.

Competing interests

The authors declare no competing interests.

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No competing interests reported.

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CPI-613 Suppresses Pancreatic Cancer Stem-Like Cells by Targeting the SEMA3A/NRP1 Pathway

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Cell Lines and Sphere Formation Assays

2.2 Colony Formation Assay

2.3 Drug Treatment

2.4 RNA isolation and quantitative real-time PCR (qRT-PCR)

2.5 Western Blotting

2.6 Tumorigenicity in Vivo Assay

2.7 Immunohistochemistry analysis

2.8 Statistics analysis

3. Results

3.1 CPI-613 induces a decrease in PDAC stem-like positive cell frequency

3.2 CPI-613 treatment induced a decreased expression of SEMA3A in CSCs

3.3 Prognostic values of SEMA3A expression in patients with pancreatic cancer

3.4 SEMA3A is critical for the maintenance of pancreatic CSC properties

3.5 CPI-613 inhibited pancreatic CSCs by decreased SEMA3A/NRP1 expression

3.6 SEMA3A recuse the inhibition of CPI-613 on pancreatic CSCs proliferation

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1