The SIX1/LDHA Axis Promotes Lactate Accumulation and Leads to NK Cell Dysfunction in Pancreatic Cancer

Background Pancreatic cancer (PC) is a malignant cancer with poor prognosis and high mortality rate. Sine oculis homeobox homolog 1 (SIX1) participates in the development of many cancers. However, the function of SIX1 in PC is not fully understood. Methods SIX1 expression was determined using immunohistochemistry in PC tissues and cell lines. Glucose consumption, lactate production, and ATP assays were used to detect the function of SIX1. PC cells and NK cells were cocultured to study the effect of SIX1 overexpression in PC cells on NK cell function. Chromatin immunoprecipitation (ChIP) assays were used to study the relationship between SIX1 and lactate dehydrogenase A (LDHA). A series of in vitro and in vivo assays were further applied to elucidate the important role of the SIX1/LDHA axis in metabolism and NK cell dysfunction in PC. Results SIX1 was significantly upregulated in PC tissue; SIX1 overexpression promoted the glycolysis capacity of PANC-1 and CFPAC-1 cells and resulted in NK cell dysfunction after the NK cells had been cultured with PC cells. LDHA inhibitor partially restored the promotion of PC caused by SIX1 overexpression. According to ChIP assays, SIX1 directly binds to the LDHA promoter region. Moreover, LDHA inhibitor and lactate transporter blocker treatment promoted the function of NK cells cocultured with PC cells. In vivo experiments yielded the same results. Conclusion The SIX1/LDHA axis promotes lactate accumulation and leads to NK cell dysfunction in PC.


Introduction
Pancreatic cancer (PC) is a malignant tumor with a high mortality rate. The incidence of PC in the United States is gradually increasing, and the five-year survival rate is only 9% [1,2]. PC metabolism is characterized by a high rate of glycolysis, which produces energy to maintain the proliferation, migration, and invasion of PC cells [3][4][5][6][7]. Moreover, studies have found that there are a variety of mechanisms mediating the effect of glycolysis on oncogenesis and tumor development [8][9][10]. Therefore, it is crucial that we have a deep understanding of the effects of glycolysis on PC.
An increasing number of studies have found that SIX1 plays an important role in tumor glycolysis, thereby promoting tumor progression [11,12]. Glycolysis in cancer tissues often leads to lactate accumulation. The increased production of lactate is related to the malignant progression of cancer and poor patient survival outcomes [13][14][15]. The relationship between the immune system and LDH is complex. LDH is released from tumors and it is one of the markers for progression in many tumors [16]. This occurs not only because glycolysis provides more energy to cancer cells but also because the lactate produced by glycolysis can directly affect the function of immune cells [17,18]. Studies have found that lactate can be a signaling molecule that plays an important role in the regulation of the tumor immune response and cell-to-cell communication [19,20]. Apart from LDH, the tumor also secretes other inflammatory mediators that have a negative effect on the cells of the immune system [21,22]. As an important part of tumor    [23,24]. NK cell dysfunction in PC is an important cause of its malignant progression [25]. Recent studies have shown that lactate can indirectly inhibit NK cell function by regulating other immunosuppressive cells (for example, by stimulating the recruitment of myeloid-derived suppressor cells (MDSCs) or by increasing the metabolic advantage of Tregs) [26,27]. However, no research has proven whether glycolysis of tumor cells can inhibit NK cell function in PC. New methods of treating tumors that target lactate production or lactate transport are continually emerging. Our research reveals the regulatory effect of the SIX1/LDHA axis on lactate and explores the negative effect of the SIX1/LDHA axis on the function of NK cells. Therefore, targeting the SIX1/LDHA axis to regulate the effect of lactate on NK cell function may become a new method of immunotherapy for PC.

Materials and Methods
2.1. Cell Culture. Four PC cell lines (PANC-1, BXPC-3, CFPAC-1, and MIAPACA-2) and the normal human pancreatic ductal cell line HPNE were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences in Shanghai, China. The cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, USA) containing 10% fetal bovine serum (Wisent, Canada) at 37°C in a humidified incubator with 95% air and 5% CO 2 . To investigate the effects of the SIX1/LDHA axis on NK cell function, 3 × 10 5 NK cells were cocultured with 5 × 10 5 PANC-1 or CFPAC-1 cells per well in 6-well plates in the presence or absence of 10 μM GNE-140 (a specific blocker for LDHA) or 10 μM 7ACC1 (a specific blocker for MCT1 and MCT4).

Patients and Pancreatic
Tissues. One hundred pairs of pancreatic tumor tissue and adjacent nontumor tissue sam-ples were obtained from patients who underwent surgery at the First Affiliated Hospital of Nanjing Medical University, China. No antitumor therapy treatment was given to the patients before surgery. Histopathological diagnoses were confirmed by two experienced pathologists. Written informed consent was obtained from the patients or their relatives, and the study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University (2021-SRFA-236).

Peripheral Blood Mononuclear Cell (PBMC) Isolation
and NK Cell Expansion. Peripheral blood was collected from healthy males with written informed consent and diluted with PBS. PBMCs were separated using the Lymphoprep density gradient centrifugation method. The isolated PBMCs were used to expand NK cells using the Natural Killer Cells Culture Kit Plus (Dakewe, China) according to the instructions. The final purity of the NK cells (CD3-, CD16+, and/ or CD56+) was >90% as quantified by flow cytometry.

Western
Blotting. The proteins were subjected to 8-12% SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, USA). The membranes were then incubated with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween (TBST) at room temperature for 2 h and incubated with primary antibodies at 4°C overnight. Following washing with TBST (15 min) three times, the membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies at room temperature for 2 h. The protein expression levels were visualized with Immobilon Western Chemilum HRP substrate (Merck Millipore, Darmstadt, Germany) using an enhanced chemiluminescence detection system. The primary antibodies against granzyme B, perforin, and MCT4 were obtained from Proteintech (Wuhan, China); the primary antibodies against LDHA, SIX1, and TNF-α were obtained from Servicebio (Wuhan, China); and the primary antibody against MCT1 was obtained from Beyotime Biotechnology (Shanghai, China).  10 20 Follow up duration (months) 30 40 Low level expression (n = 50) High level expression (n = 50) (Log-rank) (g)  Note: * P < 0:05, * * P < 0:01, and * * * P < 0:001. G1 stands for well differentiation, G2 stands for moderately differentiation, and G3 stands for poor differentiation.   Table S1.
2.8. ChIP. ChIP assays were performed using the EZ ChIP kit (Millipore, Germany) according to the manufacturer's instructions. Lysates of PANC-1 or CFPAC-1 cells were incubated with antibodies against SIX1. The probable binding motifs for SIX1 (<2000 bp proximal promoter region) were assessed using the JASPAR database (http://jaspar .genereg.net/), and the two most likely binding motifs lie within the -1902 to -1913 and -1759 to -1770 LDHA promoter regions. The length of the ChIP qPCR products containing promoter sites 1 and 2 was 188 bp and 265 bp, respectively. The primers are shown in Supplementary Table S1.
2.9. Flow Cytometric Analysis. After cocultivating NK cells and PC cells with different treatments for three days, NK cells were collected and washed twice with PBS, and then, anti-human CD3-PerCP/Cyanine5.5, CD16-PE, CD56-PE, NKG2D-APC, NKp46-PE/Cyanine7, and DNAM-1-FITC antibodies were added (BioLegend, San Diego, CA, USA) according to the instructions. The cells were incubated for 20 min in the dark and washed twice with PBS. For the detection of NK cells in mouse peripheral blood, 100 μl mouse peripheral blood was collected, and anti-mouse CD3-PerCP/Cyanine5.5, NK1.1-PE-Cyanine7, NKG2D-APC, NKp46-FITC, and DNAM-1-PE antibodies were added (BioLegend, San Diego, CA, USA) and incubated at room temperature for 20 minutes in the dark. Then, 2 ml RBC lysis solution was added to each tube, mixed well, and incubated for 15 min. Then, the samples were centrifuged, the supernatant was discarded, and the cells were washed twice with PBS. All samples were then analyzed by multicolor flow cytometry (BD, USA).

Glucose Consumption, Lactate Production, and ATP
Assays. PANC-1 and CFPAC-1 cells were seeded into 6well plates and cultured under different treatment conditions. The Glucose Assay Kit (Abcam, USA) and Lactate Assay Kit (Abcam, USA) were used to test glucose consumption and lactate production following the manufacturer's protocol, respectively. An ATP assay kit (Beyotime, China) was used to measure ATP levels according to the manufacturer's instructions. The relative levels of glucose consumption, lactate production, and ATP in all treated groups were normalized to the control group.
2.11. Animal Study. Female C57BL/6 mice (8-12 weeks old) were purchased from the Animal Center of Nanjing Medical University. PanO2 cells (1 × 10 6 cells/100 μl per flank) were injected subcutaneously into the flanks to establish tumorbearing mice. Two weeks after PanO2 cells were injected, drugs were administered by intraperitoneal injection for 2 weeks. The mice were randomly assigned to the following three groups: (1) control group, (2) anti-MCT1 and MCT4 (7ACC1, 0.3 mg/kg daily), and (3) anti-LDHA (FX-11, 2 mg/kg daily). Tumor size was measured every week for 28 days, and the formula ðwidth 2 × lengthÞ/2 was used to calculate tumor volume. Then, the mice were killed, and blood was collected for flow cytometric analysis. This study was approved by the Animal Ethical and Welfare Committee of NJMU (IACUC-1903002).    Statistical analysis in the current study was performed using GraphPad Prism (version 6.0) and SPSS software (version 22.0). Quantitative data are presented as the mean (±SD). Differences in the mean between two groups were analyzed by Student's t test. The χ 2 test was employed to analyze the associations of SIX1 or LDHA expression with clinicopathologic features. SIX1-LDHA interaction tests were performed using linear regression models. The Kaplan-Meier test was applied to calculate survival rates, and log-rank tests were used to examine differences in survival rates. P < 0:05 was considered to indicate statistical significance ( * P < 0:05, * * P < 0:01, and * * * P < 0:001).

SIX1 Promotes the Malignant Biological Behavior of PC.
SIX1 expression was detected in 100 pairs of PC and adjacent noncancerous pancreatic tissue samples by IHC. SIX1 expression was upregulated in 94 of the 100 PC samples (Figures 1(a) and 1(b)). Overall, SIX1 expression was significantly higher in PC tissues than in adjacent nonneoplastic tissues (Figures 1(c) and 1(d)). Furthermore, we examined SIX1 expression in a normal human pancreatic ductal cell line (HPNE) and PC cell lines (PANC-1, BXPC-3, CFPAC-1, and MIAPACA-2) by qRT-PCR and western blotting. As shown in Figures 1(e) and 1(f), the expression of SIX1   Then, we analyzed the correlation between SIX1 expression and the clinicopathological characteristics of PC patients. The cutoff value for high/low SIX1 expression was determined by the median expression value based on IHC scores. As presented in Table 1, no significant association was observed between the SIX1 expression levels and gender, age, location, tumor differentiation, tumor-node-metastasis (TNM) stage, T stage, N stage, or major vascular invasion, but the SIX1 expression levels correlated positively with nerve infiltration and serum CA199 values (P = 0:046 and P = 0:029, respectively). However, the Kaplan-Meier survival curves showed that SIX1 had no influence on the overall survival (OS) of PC patients (Figure 1(g)). The above findings suggested that the expression of SIX1 is upregulated in PC cells and that SIX1 promotes the malignant biological behavior of PC.

SIX1 Promotes Lactate Accumulation in PC Cells and
Leads to NK Cell Dysfunction. To further investigate the function of SIX1, PANC-1 and CFPAC-1 cells were transfected with the SIX1 overexpression vector or siRNA. The transfection efficiency was assessed by qRT-PCR and western blotting (Figures 2(a) and 2(b)). As shown in Figures 2(c) and 2(d), SIX1 overexpression significantly increased glucose uptake in PANC-1 and CFPAC-1 cells, while knocking down SIX1 showed the opposite effect. Furthermore, lactate production and ATP levels in PC cells increased after SIX1 overexpression (Figures 2(e)-2(h)).
To study the effect of SIX1 overexpression in PC cells on NK cell function, PC cells and NK cells were cocultured. As shown in Figures 3(a) and 3(b), the percentages of activating surface receptor (NKp46 and NKG2D)-positive NK cells were significantly lower after exposure to SIX1-overexpressing PANC-1 and CFPAC-1 cells, while SIX1 siRNA had the opposite effect. However, there were no significant changes in the percentages of DNAM-1-positive NK cells after the NK cells had been cultured with PANC-1 and CFPAC-1 cells. Furthermore, western blotting showed that NK cells expressed fewer cytotoxic mediators, including perforin, granzyme B, and TNF-α, after exposure to SIX1-overexpressing PANC-1 and CFPAC-1 cells, while SIX1 siRNA had the opposite effect (Figures 3(c) and 3(d)). The above results reveal that SIX1 promotes glycolysis in PC cells and that lactate accumulation results in NK cell dysfunction in PC.

LDHA Is a Functional Target of SIX1.
To identify downstream effectors of SIX1, the relationship between SIX1 and 16 glycolysis-related genes was analyzed using The Cancer Genome Atlas (TCGA) database. As shown in Figure S1, HK1, PKM2, and LDHA showed a strong correlation with SIX1 (P < 0:001, r > 0:4). We further studied the relationship between SIX1 and LDHA expressions, as measured by qRT-PCR and western blotting, SIX1 overexpression led to increased LDHA expression, and SIX1 knockdown led to decreased LDHA expression in PANC-1 and CFPAC-1 cells (Figures 4(a) and 4(b)). We then analyzed the expression of LDHA and SIX1 in PC tissues by IHC. As shown in Figure 4(c), LDHA protein expression correlated positively with SIX1 expression (P < 0:001, r = 0:58). In addition, ChIP-qPCR revealed that the LDHA promoter region in the two predicted binding sites exhibited significant enrichment after immunoprecipitation with an anti-SIX1 antibody (Figure 4(d)).

Upregulation of LDHA Is Associated with Poor Outcomes in PC.
IHC was performed to detect LDHA expression in 100 pairs of PC and adjacent nontumor pancreatic tissue samples. LDHA expression was upregulated in 91 of the 100 PC samples (Figures 5(a) and 5(b)). Overall, LDHA expression was significantly higher in PC tissues than in adjacent nonneoplastic tissues (Figures 5(c) and 5(d)).
Then, we analyzed the correlation between LDHA expression and the clinicopathological characteristics of PC patients. The cutoff value for high/low LDHA expression was the median expression value based on IHC scores. As presented in Table 1, no significant association was observed between LDHA expression levels and gender, age, location, tumor differentiation, N stage, nerve invasion, or major vascular invasion, but LDHA expression levels correlated positively with TNM stage, T stage, and serum CA199 values (P = 0:045, P = 0:001, and P = 0:029, respectively). The Kaplan-Meier survival curves showed that patients with higher LDHA expression had lower overall postoperative survival ( Figure 5(e)). In summary, our data show that 13 Journal of Immunology Research LDHA is highly expressed in PC and is closely related to clinicopathological features and prognosis.

LDHA-Mediated Lactate Production in PC Cells
Promotes NK-Cell Dysfunction. The main function of LDHA is to catalyze the formation of lactate from pyruvate. Therefore, we detected the expression of lactate transporters in PC cells and NK cells through western blotting ( Figure 5(f)). Next, we used an LDHA inhibitor (GNE-140) and a lactate transporter (MCT)-specific blocker (7ACC1) to inhibit the production and transport of lactate. As shown in Figures 6(a) and 6(b), the percentages of NKG2D-and NKp46-positive NK cells were significantly higher after PANC-1 and CFPAC-1 cells were exposed to GNE-140 and 7ACC1 (Figures 6(a) and 6(b)). Furthermore, western blotting showed that NK cells expressed more perforin, granzyme B, and TNF-α after cocultured with GNE-140and 7ACC1-treated PC cells (Figures 6(c) and 6(d)). The above results revealed that LDHA-mediated lactate production in PC cells promotes NK cell dysfunction.
3.6. LDHA Can Partially Restore the Effect of SIX1 in PC. To further investigate whether LDHA is involved in the role of SIX1 in PC, LDHA inhibitor was used to treat SIX1overexpressing PANC-1 and CFPAC-1 cells. The results showed that the increase in glycolysis capacity induced by SIX1 overexpression was recovered by the LDHA inhibitor (Figures 7(a)-7(f)). The decreased percentages of NKG2Dand NKp46-positive NK cells cocultured with SIX1overexpressing PC cells were recovered with GNE-140 treatment (Figures 7(g) and 7(h)). Furthermore, western blotting showed that NK cells cocultured with SIX1-overexpressing PC cells treated with GNE-140 expressed lower levels of cytotoxicity mediators than the SIX1-overexpressing group (Figures 7(i) and 7(j)). These results indicated that LDHA, as a functional target of SIX1, may play an important role in promoting glycolysis and NK cell dysfunction under conditions of SIX1 overexpression.

LDHA-Mediated Lactate Production Promotes NK Cell
Dysfunction In Vivo. PanO2 cells were implanted into C57BL/6 mice subcutaneously, and we measured the sizes of the xenograft tumors every 7 days for 28 days. As shown in Figures 8(a)-8(c), the tumor volumes were significantly smaller, and the tumor weights were significantly reduced after treatment with a lactate transporter blocker (7ACC1) and LDHA inhibitor (FX-11) compared with those in the control group. Furthermore, the percentages of NKG2D-, NKp46-, and DNAM-1-positive NK cells in the blood were significantly higher after intraperitoneal injection of 7ACC1 or FX-11 (Figures 8(d)-8(f)). Taken together, these results demonstrate that LDHA-mediated lactate production promotes NK cell dysfunction in PC.

Discussion
Glycolysis is a key feature of cancer, and lactate, as a product of glycolysis, plays an important role in the malignant progression of tumors [14,28]. The innovative discovery of this study is that the SIX1/LDHA axis may mediate the upstream regulatory mechanism of lactate and that lactate produced by glycolysis in PC has an inhibitory effect on NK cell function.
As a transcription factor, SIX1 participates in the development of normal tissues by regulating cell proliferation, apoptosis, or differentiation [29]. Studies have shown that the increased expression of SIX1 in tumors, such as in hepatocellular carcinoma [30], breast cancer [31], and colorectal cancer [32], often indicates poor clinical outcomes and promotes tumor proliferation, invasion, and metastasis. Recent studies have shown that SIX1 also promotes cancer progression by promoting glycolysis. Li et al.'s research shows that SIX1 directly increases the expression of many glycolytic genes, promoting the Warburg effect and tumor growth in vitro and in vivo [11]. However, whether and how SIX1 affects PC development and progression remain unclear. This study found that SIX1 expression was increased in PC tissues and cell lines. Clinical data analysis showed that PC  Journal of Immunology Research patients with high SIX1 expression had higher levels of nerve invasion and serum CA19-9 expression than those with low SIX1 expression. Moreover, SIX1 overexpression increased glucose uptake and lactate and ATP production in PC cells.
The small sample size may be the reason Kaplan-Meier survival curve analysis showed that SIX1 had no influence on the survival of PC patients.
To identify SIX1 downstream effectors, we analyzed the correlations between SIX1 and some main glycolysisrelated genes through TCGA database. We found that SIX1 is positively correlated with many main glycolysis genes and that SIX1 may be a key regulator of glycolytic gene expression. LDHA attracted our attention because it is not only a commonly used clinical test index but also shows a strong correlation with NK cell function [18,33]. In this study, we analyzed the relationship between SIX1 and LDHA. According to the immunohistochemical results of the tissue microarray, we found that LDHA expression is positively correlated with SIX1 expression. Moreover, through ChIP experiments, we found that SIX1 can directly bind to the promoter region of LDHA. Further study revealed that LDHA expression in cancer tissues of patients with PC is higher than that in adjacent tissues, and patients with high LDHA expression have a worse prognosis than those with low expression. Functional recovery experiments showed that SIX1 has a direct positive regulatory effect on LDHA. LDHA is an important enzyme in the glycolysis pathway that catalyzes the formation of lactate from pyruvate in the acidic microenvironment of cancer. Studies have shown that glycolysis has an important impact on tumor immunity. Cancer cells are more prone to glycolytic metabolism, which leads to the depletion of extracellular glucose and the release of large amounts of lactic acid, but this leads to tumor-infiltrating T cell dysfunction [34]. Similarly, pancreatic tumors that overexpress the lactate exporter MCT4 have a negative impact on the surrounding immune environment milieu [35]. These studies usually emphasize that glycolysis or lactic acid inhibits the immune function of T cells, but there are relatively few studies on NK cells.
NK cells are mainly distributed in peripheral blood, accounting for approximately 5-15% of PBMCs [36,37]. Activated NK cells can synthesize and secrete a variety of killing mediators, such as perforin, granzyme B, and TNFα, which can regulate immunity and directly kill tumor cells [38]. This study revealed a new mechanism by which glycolytic metabolism in PC affects NK cell function. In this study, we cocultured PC cells and NK cells. After SIX1 overexpression in PC cells, the percentages of activating surface receptor (NKG2D and NKp46)-positive NK cells decreased, and the secretion of perforin, granzyme B, and TNF-α decreased. 7ACC1 is a blocker of MCT1 and MCT4 and thus is a blocker of lactate transport [39,40]. In the experiment, we used 7ACC1 to inhibit MCT1 and MCT4 in a coculture system. The results showed that the function of NK cells was enhanced after 7ACC1 treatment, and the inhibitory effect of SIX1 on NK cells was partially reversed by 7ACC1. Similarly, the inhibitory effect of SIX1 on NK cells was partially restored by treatment with the LDHA inhibitor GEN140. The above results indicate that the inhibitory effect of PC on NK cells is mediated by lactate produced via the SIX1/LDHA axis. Furthermore, we also generated a PC model in mice. Flow cytometry was used to detect peripheral blood NK cells in mice treated with 7ACC1 and the LDHA inhibitor FX-11, and we found that the function of NK cells in peripheral blood was enhanced. In summary, we can conclude that lactate produced via the SIX1/LDHA axis is likely to localize to NK cells and act as a signaling molecule to mediate NK cell dysfunction. However, the specific mode of action of lactate in NK cells is still unclear and needs further study. Recently, researchers have discovered lactate-  Journal of Immunology Research derived histone lysine lactylation as a new epigenetic modification [41,42]. Whether lactate affects the function of NK cells through this pathway is also worthy of in-depth study.

Conclusion
In summary, this study comprehensively discussed the important role of the SIX1/LDHA axis in cell metabolism and NK cell dysfunction in PC and indicated that it has the potential to become a new target for the treatment of PC.

Data Availability
Please contact the author for data request.

Ethical Approval
The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was approved by the Ethics Committee of the First Affiliated Hospital with Nanjing Medical University (2021-SRFA-236) and Animal Ethical and Welfare Committee of NJMU (IACUC-1903002). (d-f) The percentages of NKG2D-, NKp46-, and DNAM-1-positive NK cells in the blood of C57BL/6 mice treated with 7ACC1 or FX-11 by intraperitoneal injection. The data are presented as the mean ± SD from three independent experiments. * P ≤ 0:05, * * P ≤ 0:01, and * * * P ≤ 0:001.