MFSD2A suppresses CD8+ T cell exhaustion in colorectal cancer by inhibiting ERK/p38 MAPK-PD-L1 signaling

Abstract

BACKGROUND

Although immune checkpoint inhibitors have shown remarkable efficacy in multiple malignancies, their clinical benefit remains limited in the majority of colorectal cancers (CRC). This resistance is attributed to CD8+ T cell exhaustion. The extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (p38 MAPK) pathway plays a pivotal role in both tumor progression and immune evasion, but its upstream regulators in CRC are unclear. Major facilitator superfamily domain containing 2A (MFSD2A), a recognized tumor suppressor and lipid transporter, has not been well characterized in terms of its immunological functions, suggesting its function as a regulator of this critical immunosuppressive axis in CRC.

AIM

To define the role of MFSD2A in CRC and elucidate its novel mechanism in alleviating CD8+ T cell exhaustion through the ERK/p38 MAPK-programmed death-ligand 1 axis.

METHODS

CRC cell lines with stable MFSD2A overexpression or knockdown were established via lentiviral transduction. In vitro assays were conducted to assess tumor cell proliferation, apoptosis, migration, and invasion. Co-culture systems incorporating CD8+ T cells were employed to evaluate cytotoxic activity, expression of exhaustion markers (programmed cell death protein 1 and cytotoxic T lymphocyte-associated antigen 4), and cytokine secretion. Epidermal growth factor was used in rescue experiments to reactivate ERK/p38 MAPK. The tumor-suppressive and immunomodulatory effects of MFSD2A were further validated in vivo using a subcutaneous tumor model.

RESULTS

MFSD2A expression was significantly downregulated in CRC cells compared with normal colonic epithelial cells. Overexpression of MFSD2A markedly inhibited proliferation, migration, and invasion while promoting apoptosis in MC38 cells, whereas MFSD2A knockdown exacerbated malignant phenotypes in Caco-2 cells. Mechanistically, MFSD2A suppressed ERK and p38 MAPK phosphorylation, reduced programmed death-ligand 1 expression in tumor cells, and decreased programmed cell death protein 1 and cytotoxic T lymphocyte-associated antigen 4 expression in co-cultured CD8+ T cells. These effects were reversed by epidermal growth factor-mediated reactivation of ERK/p38 MAPK signaling. In vivo, MFSD2A overexpression significantly inhibited tumor growth, reduced Ki67 expression, attenuated ERK/p38 MAPK activation and CD8+ T cell exhaustion, and concomitantly enhanced CD8+ T cell infiltration within the tumor microenvironment.

CONCLUSION

MFSD2A suppresses CRC progression by inhibiting ERK/p38 MAPK signaling, thereby reducing CD8+ T cell exhaustion and enhancing antitumor immunity. These findings identify MFSD2A as a promising immunotherapeutic target for CRC.

Key Words: Colorectal cancer; Major facilitator superfamily domain containing 2A; Extracellular signal-regulated kinase; p38 mitogen-activated protein kinase; T cell exhaustion

Core Tip: Major facilitator superfamily domain containing 2A, a known tumor suppressor and lipid transporter, is identified as a novel immune regulator in colorectal cancer. Major facilitator superfamily domain containing 2A exerts dual antitumor effects by suppressing the extracellular signal-regulated kinase/p38 mitogen-activated protein kinase-programmed death-ligand 1 signaling axis, thereby alleviating CD8+ T cell exhaustion and providing a potential strategy to overcome immunotherapy resistance in colorectal cancer.

INTRODUCTION

High rates of tumor recurrence, metastasis, and therapeutic resistance pose substantial challenges to colorectal cancer (CRC) management and contribute to its persistently high mortality[1-3]. CRC accounts for the second highest number of cancer deaths[4]. Although immune checkpoint inhibitors (ICIs) have achieved substantial clinical success in various cancers, their efficacy in CRC has been limited, particularly in microsatellite-stable tumors, which account for approximately 85% of CRC cases and exhibit a very low response rate to ICIs[5]. The core mechanism of this resistance lies in T cell exhaustion[6]. Prolonged exposure to tumor antigens drives sustained expression of inhibitory receptors on tumor-infiltrating CD8+ T cells, such as programmed cell death protein 1 (PD-1)[7]. These receptors act as negative immune regulators, suppressing CD8+ T cell function and promoting immune tolerance to tumors[8]. In CRC, although abundant tumor-infiltrating lymphocytes are associated with a positive prognosis, persistent immunosuppression sustains T cell exhaustion and limits effective antitumor immunity[9]. Therefore, reversing T cell exhaustion has become a key strategy to overcome immunotherapy resistance[10].

Therefore, identifying novel regulators that simultaneously modulate tumor progression and T cell exhaustion is key to improving immunotherapeutic outcomes. PD-1 is predominantly expressed on activated CD4+ and CD8+ T cells, while its ligand, programmed death-ligand 1 (PD-L1), is constitutively expressed at low levels on normal cells to maintain immune homeostasis. However, aberrant PD-L1 expression on tumor cells enables engagement with PD-1, initiating inhibitory intracellular signaling cascades that suppress antitumor responses[11]. The prevalent overexpression of PD-L1 in various malignancies is closely linked to an adverse prognosis and diminished overall survival[12,13]. PD-1/PD-L1 interaction promotes apoptosis or exhaustion of activated T cells, thereby impairing antitumor immunity[14]. Therapeutic blockade of this axis can restore T cell effector function and reinvigorate antitumor immune responses[15]. In addition to PD-1, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is another critical immune checkpoint receptor expressed on activated T cells[16-18]. Clinically, CTLA-4 inhibitors are often combined with PD-1/PD-L1 blockade to enhance antitumor immunity[19,20].

The mitogen-activated protein kinase (MAPK) signaling pathway is a fundamental regulator of cancer-related biological processes, including proliferation, differentiation, apoptosis, inflammation, and immune regulation[21]. Among MAPK family, extracellular signal-regulated kinase (ERK) and p38 MAPK signaling have emerged as key drivers of tumor growth, survival, and metastasis in multiple malignancies, including CRC[22-25]. Hyperactivation of this pathway is increasingly implicated in promoting tumor immune evasion by fostering an immune-tolerant tumor microenvironment, a key mechanism of which is the upregulation of immune checkpoint molecules[26,27]. Lou et al[28] reported that suppression of p38 MAPK activation decreased PD-L1 expression in gastric cancer cells. Despite these findings, the upstream regulatory mechanisms controlling ERK/p38 MAPK signaling in CRC and its immune microenvironment remain unclear.

Major facilitator superfamily domain containing 2A (MFSD2A) has been recognized as a potential tumor suppressor in multiple cancer types, including hepatocellular, lung, and gastric carcinomas[1,29]. MFSD2A is best known for its role in lipid transport[30]; however, its immunological function within the tumor microenvironment, particularly in CRC, remains largely unexplored. Although emerging evidence suggests that MFSD2A may restrain oncogenic signaling pathways, its role in tumor-immune interactions has not been elucidated. Based on prior evidence that suppression of ERK/p38 MAPK signaling can reverse tumor-induced immunosuppression, we hypothesize that MFSD2A acts as an endogenous negative regulator of this signaling axis in CRC. Elucidating this mechanism may provide a novel rationale for targeting MFSD2A to overcome immune resistance and improve immunotherapeutic efficacy in CRC.

MATERIALS AND METHODS

Cell cultures

Stable MFSD2A-overexpressing (oeMFSD2A) or knockdown cell lines were constructed by lentiviral transduction using constructs designed and synthesized by GenePharma (Shanghai, China). Puromycin was used for stable clone selection. CD8+ T cells were isolated from the spleens of female C57BL/6 mice (6-8 weeks old) using the MojoSort™ Mouse CD8 Naive T Cell Isolation Kit (BioLegend, CA, United States).

Western blotting

Total protein was obtained from tumor tissues or differentially treated tumor cells using RIPA lysis buffer (Beyotime, Shanghai, China), followed by protein quantification via the bicinchoninic acid assay (Beyotime, Shanghai, China). Equal amounts of protein (20 μg) were separated by sodium-dodecyl sulfate gel electrophoresis (Epizyme, Shanghai, China) and transferred onto membranes (Merck Millipore, MA, United States). The membrane was incubated with the specific antibodies: MFSD2A, p-ERK1/2, ERK1/2, p-p38 MAPK, p38 MAPK, PD-L1, PD-1, CTLA-4, and GAPDH (Proteintech, IL, United States; 1:1000), followed by HRP-conjugated secondary antibodies (Proteintech, IL, United States; 1:5000). Bands were visualized using enhanced chemiluminescence (Beyotime, Shanghai, China), and intensities were quantified for analysis.

Detection of MFSD2A expression by quantitative polymerase chain reaction

Total RNA was extracted from MC38 [overexpression negative control (oeNC), oeMFSD2A] and Caco-2 [siRNA negative control (siNC), MFSD2A-knockdown (siMFSD2A)] cells using TRIzol reagent (Beyotime, Shanghai, China). MFSD2A mRNA levels were quantified by quantitative polymerase chain reaction using SYBR Green Pro Mix (EnzyValley, Guangzhou, China). Primers were synthesized by Sangon Biotech (Shanghai, China). MFSD2A forward: 5′-CAGAATGGCTCTGTGGTGGT-3′; MFSD2A reverse: 5′-CGCACGCCTAGGATCAGAAT-3′; GAPDH forward: 5′-TGAAGC AGGCATCTGAGGG-3′; GAPDH reverse: 5′-CGAAGGTGGAAGAGTGGG AG-3′.

Cell counting kit-8 assay

Cell viability under different conditions was assessed using a cell counting kit-8 reagent (Biosharp, Hefei, China) as per the manufacturer’s instructions.

Cell apoptosis

Cell apoptosis was evaluated via the Apoptosis Detection Kit (BioLegend, CA, United States). Following trypsinization (without EDTA), cells were stained for 20 minutes with a solution containing Annexin V-fluorescein isothiocyanate and propidium iodide. Cells were analyzed by flow cytometry (CytoFLEX S, Beckman Coulter, CA, United States).

Cell killing assays

MC38 cells (control, oeNC, or oeMFSD2A) and Caco-2 cells (control, siNC, or siMFSD2A) were independently co-cultured with CD8+ T cells for 24 hours. For epidermal growth factor (EGF) treatment, tumor cells were pretreated with EGF (50 ng/mL, 48 hours) before co-culture. Cytotoxicity was determined using the Cytotoxicity LDH Assay Kit (Abcam, United Kingdom), and absorbance was measured at 450 nm. Lysis proportion (A_{E + T} - A_E - A_T)/(A_Emax - A_E) × 100%. A_{E + T}: A450 nm value for the co-incubated well, A_E: A450 nm value for the tumor cell well, A_T: A450 nm value for CD8+ T cells, A_Emax: A450 nm value for complete tumor cell lysis.

Wound healing assay

A wound healing assay was performed by creating a cross-shaped scratch in near-confluent cell monolayers. Following a 24 hours pre-incubation in basic medium, wound areas were documented at 0 and 24 hours.

Transwell assay

Cell invasion was assessed using Matrigel-coated Transwell chambers (Corning, NY, United States). After 24 hours, invaded cells were fixed, stained with crystal violet, and imaged under an inverted microscope.

Enzyme-linked immunosorbent assay

Interleukin (IL)-2, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) in cell culture supernatants were determined using enzyme-linked immunosorbent assay kits (MEIMIAN, Jiangsu, China) as per the manufacturer’s instructions.

Subcutaneous tumor models in the mouse

C57BL/6 mice were assigned to three groups (n = 6 per group, 6-8 weeks, 18-20 g) and subcutaneously injected with 5 × 10⁶ MC38 cells (control, oeNC, oeMFSD2A) into the right axillary region. Tumors were harvested after 21 days for histological and immunohistochemical analyses. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Zhejiang Center of Laboratory Animals, No. ZJCLA-IACUC-20020244.

Immunohistochemical and hematoxylin and eosin staining

Following fixation in 40 g/L paraformaldehyde and paraffin embedding, tumor sections were then subjected to hematoxylin and eosin staining. For immunohistochemistry, sections were incubated with antibodies against Ki67 or CD8 (ABclonal, Wuhan, China; 1:100), followed by appropriate secondary antibodies. Ki67 was visualized using DAB, and CD8+ T-cell infiltration was detected by immunofluorescence.

Statistical analysis

Results were shown in mean ± SD. Normality and variance homogeneity were assessed before analysis. Differences between the two groups were analyzed using unpaired t-tests, while comparisons among multiple groups were performed using one-way ANOVA. All analyses were performed via GraphPad Prism 8.

RESULTS

MFSD2A inhibits the malignant progression of CRC cells in vitro

MFSD2A, a pivotal tumor suppressor, is crucial in various malignancies, notably in lung[31], gastric[32], and hepatocellular carcinomas[33]. Nevertheless, the mechanistic basis underlying the progression of CRC is not clear. This study first evaluated MFSD2A expression in CRC cell lines (MC38, Caco-2, and SW480) and in normal colonic epithelial cells using western blotting (WB). MFSD2A was abundantly expressed in normal colonic epithelial cells but was markedly downregulated in all tested CRC cell lines. Caco-2 cells exhibited the highest expression levels, followed by SW480, whereas MC38 cells showed the lowest expression (Figure 1A). To investigate the role of MFSD2A in CRC, Caco-2 and MC38 cells were selected for further investigation. OeMFSD2A MC38 cell lines and siMFSD2A Caco-2 cell lines were generated via lentiviral transduction. Successful establishment of oeMFSD2A and siMFSD2A cell models was confirmed in both WB and quantitative polymerase chain reaction analysis (Figure 1B and C). Functional assays revealed that MFSD2A overexpression markedly suppressed the proliferation of MC38 cells, whereas MFSD2A knockdown markedly promoted Caco-2 cell proliferation (Figure 1D). Furthermore, MFSD2A overexpression induced apoptosis in MC38 cells, while MFSD2A silencing suppressed apoptosis in Caco-2 cells (Figure 1E and F). The influence of MFSD2A on migratory capacity was subsequently examined. Wound-healing and Transwell assays demonstrated that oeMFSD2A markedly attenuated the migration and invasion of MC38 cells. Conversely, silencing MFSD2A in Caco-2 cells enhanced these malignant phenotypes in contrast to controls (Figure 1G, H, I, and J). These in vitro results demonstrate that MFSD2A suppresses CRC progression by inhibiting cell proliferation, migration, and invasion.

Figure 1 Major facilitator superfamily domain containing 2A inhibits the malignant progression of colorectal cancer cells in vitro. A: Western blotting analysis of major facilitator superfamily domain containing 2A (MFSD2A) expression in normal colonic epithelial cells, Caco-2, SW480, and MC38 cells; B: Validation of MFSD2A overexpression and knockdown by western blotting; C: The relative MFSD2A expression level in cells was determined by quantitative polymerase chain reaction; D: Cell proliferation was determined using the cell counting kit-8 assay; E and F: Apoptosis detected using allophycocyanin Annexin V/propidium iodide flow cytometry; G and H: Wound healing assays were performed to evaluate the migration capability of MFSD2A-overexpressing MC38 and MFSD2A-knockdown Caco-2 cells; I and J: Transwell assays were performed to evaluate the invasion capability of MFSD2A-overexpressing MC38 and MFSD2A-knockdown Caco-2 cells. ^aP < 0.0001 vs overexpression negative control, ^bP < 0.01 vs overexpression negative control, ^cP < 0.0001 vs siRNA negative control, and ^dP < 0.01 vs siRNA negative control. MFSD2A: Major facilitator superfamily domain containing 2A; NCM460: Normal colonic epithelial cells; oeMFSD2A: Major facilitator superfamily domain containing 2A-overexpressing; oeNC: Overexpression negative control; siMFSD2A: Major facilitator superfamily domain containing 2A-knockdown; siNC: SiRNA negative control.

MFSD2A suppresses the ERK/p38 MAPK-PD-L1 axis to alleviates CD8+ T cell exhaustion

Whether MFSD2A regulates ERK/p38 MAPK signaling and immune checkpoint expression in CRC cells was subsequently examined. WB analysis showed that MFSD2A overexpression in MC38 cells markedly reduced ERK and p38 MAPK phosphorylation, whereas MFSD2A knockdown in Caco-2 cells produced the opposite effect (Figure 2A). Consistently, expression of the downstream immune checkpoint PD-L1 was decreased in oeMFSD2A cells and increased in siMFSD2A cells (Figure 2A).

Figure 2 Major facilitator superfamily domain containing 2A modulates the extracellular signal-regulated kinase/p38 mitogen-activated protein kinase-programmed death-ligand 1 axis and alleviates CD8+ T cell exhaustion. A: Western blotting analysis of extracellular signal-regulated kinase/p38 mitogen-activated protein kinase pathway and programmed death-ligand 1 expression in MC38 [overexpression negative control (oeNC), major facilitator superfamily domain containing 2A-overexpressing (oeMFSD2A)] and Caco-2 [siRNA negative control (siNC), major facilitator superfamily domain containing 2A-knockdown (siMFSD2A)] cells; B: The expression of programmed cell death protein 1 and cytotoxic T lymphocyte-associated antigen 4 in CD8+ T cells following co-culture of MC38 (control, oeNC, oeMFSD2A) cells or Caco-2 (control, siNC, siMFSD2A) cells with CD8+ T cells; C: Killing assay (24 hours) showing the lysis proportion of MC38 (control, oeNC, oeMFSD2A) cells and Caco-2 (control, siNC, siMFSD2A) cells co-cultured with CD8+ T cells, respectively; D: Following co-culture of MC38 cells (control group, oeNC group, oeMFSD2A group) or Caco-2 cells (control group, siNC group, siMFSD2A group) with CD8+ T cells, enzyme-linked immunosorbent assay assays were performed to detect the interleukin-2, interferon-γ, and tumor necrosis factor-α secreted by CD8+ T cells. ^aP < 0.0001 vs overexpression negative control, ^bP < 0.0001 vs siRNA negative control. oeMFSD2A: Major facilitator superfamily domain containing 2A-overexpressing; oeNC: Overexpression negative control; siMFSD2A: Major facilitator superfamily domain containing 2A-knockdown; siNC: SiRNA negative control; ERK: Extracellular signal-regulated kinase; MAPK: Mitogen-activated protein kinase; PD-L1: Programmed death-ligand 1; CTLA4: Cytotoxic T lymphocyte-associated antigen 4; IL-2: Interleukin-2; IFN-γ: Interferon-γ; TNF-α: Tumor necrosis factor-α.

To evaluate the immunological consequences of MFSD2A-modulated tumor cells, a tumor cell-CD8+ T cell co-culture system was employed. Compared with the oeNC MC38 group, CD8+ T cells co-cultured with oeMFSD2A MC38 cells exhibited significantly reduced expression of the exhaustion markers PD-1 and CTLA-4 (Figure 2B). In contrast, CD8+ T cells co-cultured with siMFSD2A Caco-2 cells displayed markedly elevated levels of these inhibitory receptors relative to the siNC group (Figure 2B). Functionally, these phenotypic changes were accompanied by enhanced cytotoxic activity of CD8+ T cells against oeMFSD2A MC38 cells and diminished killing capacity against siMFSD2A Caco-2 cells (Figure 2C). In parallel, secretion of the IL-2, IFN-γ, and TNF-α was significantly increased in CD8+ T cells co-cultured with oeMFSD2A cells, but decreased following co-culture with MFSD2A-silenced cells (Figure 2D).

This study identifies MFSD2A as a critical negative regulator of the oncogenic ERK/p38 MAPK-PD-L1 axis in CRC cells. By attenuating this signaling pathway, MFSD2A not only suppresses tumor-intrinsic oncogenic signaling but also alleviates CD8+ T cell exhaustion. This mechanistic link prompted further validation through rescue experiments.

MFSD2A suppresses malignant phenotypes and PD-L1 expression in CRC cells via the ERK/p38 MAPK pathway

To determine whether the tumor-suppressive effects of MFSD2A are mediated through the ERK/p38 MAPK pathway, oeMFSD2A MC38 cells were treated with EGF (50 ng/mL), a potent activator of this signaling axis. EGF treatment effectively reactivated ERK and p38 MAPK signaling, as evidenced by a marked increase in their phosphorylation levels (Figure 3A). Consistent with this reactivation, EGF significantly abrogated the suppressive effects of MFSD2A, restoring cell proliferation (Figure 3B), reducing apoptosis (Figure 3C), and enhancing migratory and invasive capacities (Figure 3D and E) compared with untreated oeMFSD2A cells. Notably, reactivation of ERK/p38 MAPK signaling also resulted in an upregulation of PD-L1 expression (Figure 3F), providing a mechanistic link between MFSD2A-mediated MAPK inhibition and immune checkpoint regulation. Collectively, these rescue experiments demonstrate that MFSD2A suppresses malignant phenotypes in CRC cells primarily through inhibition of the ERK/p38 MAPK pathway, with concomitant downregulation of PD-L1 as a downstream consequence.

Figure 3 Major facilitator superfamily domain containing 2A suppresses malignant phenotypes and programmed death-ligand 1 expression in colorectal cancer cells via the extracellular signal-regulated kinase/p38 mitogen-activated protein kinase pathway. A: Western blotting analysis of major facilitator superfamily domain containing 2A (MFSD2A), p-extracellular signal-regulated kinase 1/2, extracellular signal-regulated kinase 1/2, p-p38 mitogen-activated protein kinase, and p38 mitogen-activated protein kinase expression in MFSD2A-overexpressing MC38 cells treated with or without epidermal growth factor (EGF); B: Cell counting kit-8 assay was performed on MC38 cells with or without EGF (50 ng/mL) treatment; C: Apoptosis assay was performed on MC38 cells with or without EGF (50 ng/mL) treatment; D: Wound healing assay was used to assess the effects of EGF on MC38 cell migration; E: Transwell assay was used to assess the effects of EGF on MC38 cell invasion; F: Western blotting analysis of programmed death-ligand 1 expression in MFSD2A-overexpressing MC38 cells treated with or without EGF (50 ng/mL). ^aP < 0.0001 vs control, ^bP < 0.0001 vs major facilitator superfamily domain containing 2A-overexpressing, ^cP < 0.01 vs major facilitator superfamily domain containing 2A-overexpressing, and ^dP < 0.001 vs Major facilitator superfamily domain containing 2A-overexpressing. oeMFSD2A: Major facilitator superfamily domain containing 2A-overexpressing; MFSD2A: Major facilitator superfamily domain containing 2A; ERK: Extracellular signal-regulated kinase; MAPK: Mitogen-activated protein kinase; EGF: Epidermal growth factor; PD-L1: Programmed death-ligand 1.

MFSD2A suppresses CD8+ T cell exhaustion via the ERK/p38 MAPK signaling pathway

To determine if the MAPK pathway mediates MFSD2A’s immunomodulatory function, we introduced EGF to the co-culture system. EGF treatment effectively reversed the T cell-enhancing effects of MFSD2A overexpression. Specifically, it significantly upregulated the exhaustion markers PD-1 and CTLA-4 on CD8+ T cells (Figure 4A). Consistently, reactivation of MAPK signaling abolished the functional benefits of MFSD2A, as evidenced by reduced CD8+ T cell cytotoxicity (Figure 4B) and decreased secretion of the effector cytokines IL-2, IFN-γ, and TNF-α (Figure 4C). Collectively, these rescue experiments demonstrate that MFSD2A alleviates CD8+ T cell exhaustion predominantly through suppression of the ERK/p38 MAPK pathway.

Figure 4 Major facilitator superfamily domain containing 2A suppresses CD8+ T cell exhaustion via the extracellular signal-regulated kinase/p38 mitogen-activated protein kinase signaling pathway. A: Western blotting analysis of programmed cell death protein 1 and cytotoxic T lymphocyte-associated antigen 4 expression in CD8+ T cells following co-culture with MC38 cells treated with or without epidermal growth factor (EGF); B: Killing assay (24 hours) showing lysis rates of major facilitator superfamily domain containing 2A-overexpressing MC38 cells co-cultured with CD8+ T cells in the presence or absence of EGF; C: Enzyme-linked immunosorbent assay quantification of interleukin-2, interferon-γ, and tumor necrosis factor-α secretion by CD8+ T cells incubated with major facilitator superfamily domain containing 2A-overexpressing MC38 cells under EGF-treated or untreated conditions. ^aP < 0.0001 vs major facilitator superfamily domain containing 2A-overexpressing. oeMFSD2A: Major facilitator superfamily domain containing 2A-overexpressing; EGF: Epidermal growth factor; PD1: Programmed cell death protein 1; CTLA4: Cytotoxic T lymphocyte-associated antigen 4; IL-2: Interleukin-2; IFN-γ: Interferon-γ; TNF-α: Tumor necrosis factor-α.

MFSD2A overexpression inhibits tumor growth and promotes CD8+ T cell infiltration, concurrent with suppressed MAPK signaling in vivo

For in vivo evaluation of MFSD2A function, a subcutaneous tumor model was established in C57BL/6 mice. During the model establishment, three mice in the MC38 and MC38 + oeNC group died. It is speculated that this may be related to the strong in vivo tumor invasiveness of this cell line at the inoculated dose. MFSD2A overexpression markedly suppressed tumor growth compared with both the oeNC and MC38 control groups (Figure 5A). Histological and immunohistochemical analyses further supported this observation, revealing looser stromal architecture and a pronounced reduction in Ki67 expression in oeMFSD2A tumors, indicative of impaired proliferative activity (Figure 5B and C). Mechanistically, tumor growth inhibition was associated with attenuation of ERK/p38 MAPK signaling, as demonstrated by significantly decreased phosphorylation of ERK and p38 in oeMFSD2A tumors (Figure 5D). Importantly, MFSD2A overexpression also reshaped the tumor immune microenvironment, as evidenced by a substantial increase in CD8+ T cell infiltration within tumors (Figure 5E). Collectively, these in vivo findings identify MFSD2A restrains tumor growth by simultaneously suppressing oncogenic ERK/p38 MAPK signaling and enhancing antitumor immune surveillance.

Figure 5 Major facilitator superfamily domain containing 2A overexpression inhibits tumor growth and promotes CD8+ T cell infiltration, concurrent with suppressed mitogen-activated protein kinase signaling in vivo. A: Subcutaneous tumor growth following injection of MC38, overexpression negative control MC38, or major facilitator superfamily domain containing 2A-overexpressing MC38 cells in C57BL/6 mice; B: Hematoxylin and eosin staining; C: Ki67 immunohistochemistry of tumor tissue; D: Western blotting analysis of p-extracellular signal-regulated kinase 1/2, extracellular signal-regulated kinase 1/2, p-p38 mitogen-activated protein kinase, and p38 mitogen-activated protein kinase expression in tumor tissue; E: Immunofluorescence staining of CD8+ T cell infiltration in tumor tissue. oeMFSD2A: Major facilitator superfamily domain containing 2A-overexpressing; oeNC: Overexpression negative control; HE: Hematoxylin and eosin; ERK: Extracellular signal-regulated kinase; MAPK: Mitogen-activated protein kinase.

DISCUSSION

In terms of global incidence, CRC ranks third among all malignancies[34,35]. Therefore, the identification of novel targets for CRC metastasis and the elucidation of mechanisms underlying CRC progression are of paramount importance for therapeutic advancement. The high metastatic potential and frequent emergence of therapeutic resistance, particularly in microsatellite-stable CRC, which is largely refractory to immune checkpoint blockade, remain major clinical challenges[36,37]. These limitations are primarily attributable to an immunosuppressive tumor microenvironment characterized by dysfunctional and exhausted CD8+ T cells. In this context, the present study identifies the lipid transporter MFSD2A as a previously unrecognized regulator that simultaneously restrains tumorigenesis and alleviates T cell exhaustion in CRC, predominantly through suppression of the ERK/p38 MAPK signaling axis.

MFSD2A is well established for its essential roles in lipid transport[38,39]. Emerging evidence, however, suggests that MFSD2A also exerts multifaceted functions in tumor biology. In lung cancer, MFSD2A suppresses tumor growth by inducing cell-cycle arrest and impairing cell-matrix adhesion[31], whereas in gastric cancer, it remodels the immune microenvironment[40]. Furthermore, high MFSD2A expression is associated with better prognosis in gastric cancer and hepatocellular carcinoma, suggesting its value as a prognostic indicator and a therapeutic target[32,33,41]. The function of MFSD2A in CRC, particularly in regulating tumor-immune interactions, has remained largely unexplored. The present study demonstrates that MFSD2A expression is markedly reduced in CRC cell lines, prompting a comprehensive investigation into its functional and mechanistic significance in CRC progression.

A key and novel contribution of this work lies in elucidating the immunomodulatory role of MFSD2A and mechanistically linking its tumor-suppressive function to inhibition of the oncogenic ERK/p38 MAPK pathway. This signaling cascade is a central driver of tumor cell proliferation, survival, and metastasis, and it also promotes immune evasion through upregulation of PD-L1[42,43]. PD-1/PD-L1 and CTLA-4 are core mediators of immune escape, and their sustained activation induces T cell exhaustion, characterized by diminished cytotoxicity and impaired effector function[44]. Our study elucidated that MFSD2A suppresses ERK and p38 MAPK phosphorylation, leading to downregulation of PD-L1 on tumor cells. This, in turn, profoundly influences CD8+ T cell function: Exhaustion markers PD-1 and CTLA-4 are reduced, cytotoxic activity is enhanced, and secretion of effector cytokines, including IL-2, IFN-γ, and TNF-α, is increased, collectively reversing the exhausted T cell phenotype.

The specificity of this mechanism was corroborated through rescue experiments. Administration of EGF, a potent activator of the MAPK pathway, effectively abrogated the suppressive effects of MFSD2A overexpression, restoring the capacities of tumor cells for proliferation and migration while reversing its enhancing impact on CD8+ T cell function. These results not only reinforce the causal relationship between MFSD2A and MAPK signaling but also underscore the therapeutic potential of targeting this axis to remodel the tumor immune microenvironment. The tumor-suppressive effect of MFSD2A was also confirmed in vivo, where its overexpression significantly inhibited tumor growth in a subcutaneous tumor model. Mechanistically, MFSD2A enhanced tumor immunogenicity, promoted infiltration of cytotoxic CD8+ T cells, and shifted the tumor microenvironment toward a more immune-permissive state. While the present in vivo validation was based on a gain-of-function approach, future studies employing MFSD2A loss-of-function models will be essential to fully elucidate its physiological and pathophysiological roles in CRC and immunotherapy responsiveness. Moreover, although MFSD2A has previously been shown to suppress CRC progression via the S100A14/STAT3 axis[1], the current study reveals a parallel and previously unrecognized mechanism involving the ERK/p38 MAPK-PD-L1 axis. Elucidating the potential crosstalk or hierarchical regulation between these pathways represents an important direction for future investigation.

CONCLUSION

MFSD2A is a pivotal tumor suppressor in CRC that operates through a dual mechanism: Intrinsically inhibiting oncogenic ERK/p38 MAPK signaling and extrinsically reinvigorating anti-tumor immunity by disrupting the ERK/p38 MAPK-PD-L1 axis. These findings provide mechanistic insight into the poor immunotherapeutic responsiveness observed in CRC patients with low MFSD2A expression. Our data offer compelling preclinical evidence that enhancing MFSD2A activity may represent a viable approach to overcoming immunotherapy resistance in CRC, particularly by modulating the immunosuppressive tumor microenvironment and improving responses to PD-1/PD-L1 blockade. Further studies are warranted to explore the translational potential of MFSD2A-targeted interventions, especially in combination with existing ICIs.