Polygonum cuspidatum Extract (Pc-Ex) Containing Emodin Suppresses Lung Cancer-Induced Cachexia by Suppressing TCF4/TWIST1 Complex-Induced PTHrP Expression

Cachexia, which is characterised by the wasting of fat and skeletal muscles, is the most common risk factor for increased mortality rates among patients with advanced lung cancer. PTHLH (parathyroid hormone-like hormone) is reported to be involved in the pathogenesis of cancer cachexia. However, the molecular mechanisms underlying the regulation of PTHLH expression and the inhibitors of PTHLH have not yet been identified. The PTHLH mRNA levels were measured using quantitative real-time polymerase chain reaction, while the PTHrP (parathyroid hormone-related protein) expression levels were measured using Western blotting and enzyme-linked immunosorbent assay. The interaction between TCF4 (Transcription Factor 4) and TWIST1 and the binding of the TCF4–TWIST1 complex to the PTHLH promoter were analysed using co-immunoprecipitation and chromatin immunoprecipitation. The results of the mammalian two-hybrid luciferase assay revealed that emodin inhibited TCF4–TWIST1 interaction. The effects of Polygonum cuspidatum extract (Pc-Ex), which contains emodin, on cachexia were investigated in vivo using A549 tumour-bearing mice. Ectopic expression of TCF4 upregulated PTHLH expression. Conversely, TCF4 knockdown downregulated PTHLH expression in lung cancer cells. The expression of PTHLH was upregulated in cells ectopically co-expressing TCF4 and TWIST1 when compared with that in cells expressing TCF4 or TWIST1 alone. Emodin inhibited the interaction between TCF4 and TWIST1 and consequently suppressed the TCF4/TWIST1 complex-induced upregulated mRNA and protein levels of PTHLH and PTHrP. Meanwhile, emodin-containing Pc-Ex significantly alleviated skeletal muscle atrophy and downregulated fat browning-related genes in A549 tumour-bearing mice. Emodin-containing Pc-Ex exerted therapeutic effects on lung cancer-associated cachexia by inhibiting TCF4/TWIST1 complex-induced PTHrP expression.


Introduction
Lung cancer is the most lethal cancer type threatening human life and health. Approximately 22% of lung cancer-related deaths are attributed to cancer cachexia [1]. Cancer cachexia, which is a metabolic syndrome characterised by loss of body weight and decreased skeletal muscle mass, food intake (anorexia), and white adipose tissue (WAT) weight, decreases the quality of life in 60-80% of patients with cancer [2]. The wasting of skeletal muscles, a symptom of cancer cachexia, results from the dysregulation of protein synthesis and degradation in the skeletal muscles [3]. The expression levels of proteolysisrelated genes such as Trim63 and Fbxo32 (also known as Atrogin1) are upregulated in the lung cancer-induced cachectic muscle [4]. Metabolic imbalances in the skeletal muscle are mediated by tumour-derived pro-inflammatory cytokines, such as tumour necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-6, and transforming growth factor β1 (TGFβ1) [5]. These cytokines are reported to activate proteolysis and suppress protein synthesis through the activation of nuclear factor-κB (NF-κB), JAK/STAT, and Smad3 in the skeletal muscles [6,7]. Consistently, the serum levels of pro-inflammatory cytokines in patients with cachectic cancer are upregulated when compared with those in weight-stable counterparts [8]. A common feature of cancer cachexia is the loss of fat mass, which can be attributed to decreased lipid storage and lipogenesis and increased lipolysis and thermogenesis in WAT [9]. In patients with advanced cancer, WAT is converted into brown adipose tissue (BAT), which has large numbers of mitochondria and exhibits upregulated levels of thermogenic uncoupling proteins. Fat browning contributes to high energy expenditure in cancer cachexia [10]. Several studies have demonstrated that the monoclonal antibody-mediated neutralisation of IL-1 or IL-6 ameliorates cancer cachexia characterised by loss of skeletal muscle mass and WAT browning [9]. Additionally, the pharmacological inhibition of β3-adrenergic signalling is reported to decrease WAT browning and ameliorate cancer cachexia severity [11]. Many efforts have been made to evaluate biomarkers of cancer cachexia. Metabolic alterations such as amino acids and lipoproteins were occurred in the early stage of cancer cachexia, and cytokines were also altered in the cachexia stage [12,13]. Several studies have examined the therapeutic strategies for cancer cachexia by targeting biomarkers of cancer cachexia. However, therapeutics for the clinical treatment of patients with cancer cachexia are not currently available.
PTHLH, which is encoded by parathyroid hormone-related protein (PTHrP), serves as an external stimulus to maintain bone turnover and skeletal homeostasis [14]. Previous studies have reported that PTHrP promotes the growth, metastasis, and chemoresistance of various cancers, such as lung, colorectal, prostate, pancreatic, and breast cancers [15][16][17][18][19][20]. A recent study reported that tumour-derived PTHrP causes cancer cachexia by promoting WAT browning and skeletal muscle loss [4]. Activated Smad3 promotes the transcription of PTHLH in response to TGFβ1, which is also known as a cachexia-inducing factor, in breast cancer [7,17]. Monoclonal antibodies against PTHrP are reported to exert therapeutic effects on breast cancer bone metastasis and lung cancer-driven cachexia [4,15]. These findings suggest that PTHrP is a druggable target for cachexia, as well as for bone metastasis in patients with cancer. However, the mechanism underlying the regulation of PTHrP expression has not been completely elucidated.
This study demonstrated the molecular mechanisms underlying the TCF4/TWIST1 interaction-mediated PTHrP expression in lung cancer-induced cachexia.

Quantitative Real-Time PCR (qRT-PCR)
To measure mRNA expression, total RNA in cultured lung cancer cells was prepared by using TRIzol (Invitrogen, Carlsbad, CA, USA) and isopropanol (Sigma-Aldrich, St. Louis, MO, USA). To measure mRNA expression in A549-driven tumours, gastrocnemius muscle and epididymal white adipose tissue (eWAT), sacrificed tissues were frozen with liquid nitrogen and frozen tissues were pulverized by using dry ice and liquid nitrogen. Frozen tissues (30~40 mg) were homogenized with 1 mL of TRIzol (Invitrogen, Carlsbad, CA, USA) by using a bead homogenizer (Benchmark Scientific, Sayreville, NJ, USA) to extract total RNA. The purity of RNA was assessed by using a spectrophotometer (BioTek, Winooski, VT, USA) at 260 and 280 nm and a ratio of~1.8 is accepted for measurement of gene expression. The reverse transcriptase and cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA) was used for cDNA synthesis. SYBR Green qPCR mixture (Applied Biosystems, Foster City, CA, USA) was used for qRT-PCR and experimental Ct values were normalized and calculated to 36B4 (ribosomal protein subunit P0, RPLP0) or β-actin gene. Detailed primer sequences for qRT-PCR are described in Table 1.

Mammalian Two-Hybrid Luciferase Assay
pGL4.31-Luc (luc2P/GAL4UAS/Hygro) vector was used to measure VP16-TCF4 and GAL4-TWIST1 interaction-mediated luciferase expression. To perform high-throughput screening (HTS) by using 501 natural compounds, HEK293T cells were transiently transfected with pGL4.31-Luc, pFN10A-TCF4 and pFN11A-TWIST1, and then transfected cells were further incubated for 24 h to allow stabilization. Twenty-four hours post-transfection, cells were divided into 96-well cell culture plates and incubated for 24 h. Then, 5 µg/mL of natural compounds was treated in transfected cells and incubated for 24 h. To measure luciferase activities, cell lysates were reacted with luciferase assay buffer and luciferin substrate, and then luciferase activities were measured by using Luminometer (BioTek, Winooski, VT, USA).

Animal Experiment
The animal experiments were approved and performed in accordance with the guidelines of Konkuk University Institutional Animal Care and Committee (KU20062). A549 lung cancer cells (1 × 10 6 ) suspensions were prepared in FBS and antibiotics-free RPMI1640: BD Matrigel TM (BD Biosciences, Franklin Lakes, NJ, USA) mixture (100 µL) on ice and subcutaneously injected into the flank of Balb/c-nude mice. The mice were received normal animal diets (AIN93G) until a tumour size of 100 mm 3 was reached. After the tumour size reached 100 mm 3 , the mice were divided into two groups and received daily doses of AIN93G or 2% Pc-Ex in their feed for 46 days. Pc-Ex-containing animal diets were formulated depending on nutrients of Pc-Ex such as carbohydrate, protein, lipid, and minerals in accordance with AIN93G ( Table 3). The bodyweight in all of the mice was measured once a week. Tumour volumes were measured by using calipers once a week and calculated from the following equation: volume = ab 2 /2, where a is the maximal width and b is the maximal orthogonal width. When animal experiments were terminated, gastrocnemius muscle, eWAT and tumours were harvested, and all of the harvested tissues were frozen with liquid nitrogen. LLC1 cells were seeded in a 12-well plate (1 × 10 4 per well) and incubated with Pc-Ex (100 µg/mL) for 24 h in the presence of TGFβ1 (20 ng/mL). The supernatants of cells were collected and the PTHrP protein levels were measured using mouse ELISA (Enzyme-linked Immunosorbent Assay). All procedures were performed according to the manufacturer's instructions (AVIVA SYSTEMS BIOLOGY, San Diego, CA, USA).

Preparation of Pc-Ex and Measurement of Emodin Concentration
For the preparation of Polygonum cuspidatum extracts, the powder of a dried Polygonum cuspidatum was obtained from the Jung-Ang microbe research institute (Cheongju, Korea). The powder was then immersed in water, sonicated for 15 min, and extracted for 24 h. The extract was filtered through non-fluorescent cotton and evaporated under reduced pressure by using a rotary evaporator at 40 • C. The condensed extract was then lyophilized by using a Modul Spin 40 dryer (Biotron Corporation, Calgary, AB, Canada) for 72 h. The plant extracts were stored at −20 • C and reconstructed with 50 mL of methanol before the HPLC analysis. All of the Polygonum cuspidatum extract samples were milled into powders, and an individual portion of the powdered samples (0.2 g) was dissolved in methanol. The resulting samples were then extracted for 15 min using an ultrasonic cleaner in a water bath (30 • C). The extracts were filtered by a GHP syringe filter and the elution was injected directly into the HPLC system for the analysis. Emodin (30269, ≥97) and formic acid (F0607, ≥95%) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Methanol, Water (HPLC-grade) was obtained from Fisher Scientific Co.(Fair Lawn, MA, USA). Double-distilled water was obtained using a Millipore Milli-Q Plus water treatment system (Millipore, Burlington, MA, USA). The stock solutions of five standards were made at a concentration of 10.3 mg in a final volume of 100 mL of methanol. Working solutions of mixed standards at the concentrations of 7.1, 14.1, 28.3, 56.5, and 113.0 µg were made by dilution of stock solution in volumetric flasks with the mobile phase. Then the standards were injected into the HPLC. High-performance liquid chromatography 20 µL samples were analyzed on a Zorbax Eclipse XDB-C18 column (4.6 × 250 mm, 5 µm, Agilent, Santa Clara, CA, USA), which was maintained at 40 • C using an Agilent Infinity-1260 HPLC system. The mobile phases consisted of (A) water containing 0.1% (w/w) formic acid and (B) methanol. The HPLC elution conditions were optimized as follows: linear gradient from 20 to 35% B (0 to 13 min), 35 to 100% B (13 to 30 min), and 100 to 20% B (30 to 40 min), where it was held for 3 min. The flow rate was set at 1.0 mL/min, and the column and autosampler were maintained at 30 and 25 • C, respectively. The scan range for the DAD detector system was set at 190 to 400 nm. Analogue output channel A was set at wavelength 254 nm with a bandwidth of 4 nm. The injection volume was 20 µL.

PTHLH Expression Is Upregulated in Response to TGFβ1 in Lung Cancer
PTHLH is correlated with the pathogenesis of cancer cachexia, which is characterised by WAT browning, skeletal muscle loss, and bone metastasis [4,17]. In this study, the expression levels of PTHLH mRNA in patients with lung cancer were investigated using publicly available gene expression datasets. Bioinformatic analysis of GSE74706 [33] and GSE22863 datasets [34] revealed that the PTHLH mRNA levels were upregulated in nonsmall cell lung cancer (NSCLC) and primary lung cancer ( Figure 1A). The TGFβ1 signalling pathway has a critical role in the biological processes of metazoans. The dysregulation of the TGFβ1 signalling pathway leads to tumour development and metastasis through the induction of EMT [35]. A recent study reported that the TGFβ1 signalling pathway upregulates the expression of PTHLH in lung and breast cancers [36]. Consistent with the findings of previous studies, the expression of PTHLH was significantly upregulated in TGFβ1-treated A549, NCI-H1299, NCI-H1650, NCI-H358, L132, Calu-1, and Calu-3 lung cancer cells ( Figure 1B). Through bioinformatic analysis of the GSE114761 dataset [37] increased PTHLH mRNA levels were found in TGFβ1-treated many types of lung cancer cells ( Figure 1C). These results indicate that PTHLH may be associated with TGFβ1-mediated lung cancer aggressiveness.
Nutrients 2022, 14, x FOR PEER REVIEW 7 of 20 cells ( Figure 1C). These results indicate that PTHLH may be associated with TGFβ1-mediated lung cancer aggressiveness.

TCF4 Regulates PTHLH Expression in Lung Cancer Cells
Previous studies have reported that the TGFβ1 signalling pathway promotes the interaction between TCF4 and TWIST1 and consequently upregulates the expression of

TCF4 Regulates PTHLH Expression in Lung Cancer Cells
Previous studies have reported that the TGFβ1 signalling pathway promotes the interaction between TCF4 and TWIST1 and consequently upregulates the expression of EMTrelated genes [16]. The interaction between TCF4 and TWIST1 was also confirmed in this study (Figure 2A). TCF4 interacted with the bHLH domain of TWIST1 ( Figure 2B). The expression of PTHLH is upregulated in TGFβ1-treated breast and lung cancer cells [17,[38][39][40]. Thus, we hypothesized that TCF4 functions as an essential transcription factor to regulate PTHLH expression in response to TGFβ1 signalling. Adenoviral transduction of TCF4 upregulated PTHLH mRNA expression in A549, Calu-1, and NCI-H358 epithelial lung cancer cells ( Figure 2C). The efficiency of adenoviral infection was examined by measuring the TCF4 protein levels ( Figure 2D). The PTHLH mRNA levels were upregulated in lung cancer cells ectopically overexpressing Myc-TCF4 ( Figure 2E). TGFβ1-induced PTHLH expression was mitigated in TCF4-silenced A549 ( Figure 2F) and Calu-1 lung cancer cells ( Figure 2G). These results indicate that TCF4 is involved in TGFβ1-induced PTHLH expression in lung cancer cells. To examine the functional role of TCF4 and TWIST1 in regulating PTHLH expression, the PTHLH mRNA levels were measured in lung cancer cells co-expressing TCF4 and TWIST1. The PTHLH and EMT-promoting SNAI2 mRNA levels in the cells ectopically overexpressing TCF4 or TWIST1 alone was approximately twofold higher than those in cells infected and transfected with green fluorescence protein (GFP)-expressing adenovirus and empty vector ( Figure 2H,I). Compared with those in the infected and transfected with GFP-expressing adenovirus and empty vector, the PTHLH and SNAI2 mRNA levels were upregulated by approximately sevenfold (A549) or fourfold (NCI-H358) in cells co-expressing TCF4 and TWIST1 ( Figure 2H,I). These results indicate that cooperation between TCF4 and TWIST1 promotes transcription of PTHLH and the expression of EMT-related genes, such as SNAI2 in lung cancer cells.
x FOR PEER REVIEW 8 of 20 approximately twofold higher than those in cells infected and transfected with green fluorescence protein (GFP)-expressing adenovirus and empty vector ( Figure 2H,I). Compared with those in the infected and transfected with GFP-expressing adenovirus and empty vector, the PTHLH and SNAI2 mRNA levels were upregulated by approximately sevenfold (A549) or fourfold (NCI-H358) in cells co-expressing TCF4 and TWIST1 ( Figure  2H,I). These results indicate that cooperation between TCF4 and TWIST1 promotes transcription of PTHLH and the expression of EMT-related genes, such as SNAI2 in lung cancer cells.   The interaction between TCF4 and TWIST1 promotes lung cancer growth, metastasis, and cachexia by upregulating the expression of PTHLH and EMT-related genes. This indicated that this transcriptional axis is a potential druggable target for lung cancer growth, metastasis, and cachexia. The mammalian two-hybrid luciferase system is based on protein-protein interaction (PPI)-mediated luciferase expression. This system comprises GAL4-fusion and VP16-fusion proteins and is widely used to identify PPI inhibitors. GAL4tagged TWIST1 and VP16-tagged N-terminal (N, amino acids 1-250), middle domain (M, amino acids 251-500), and C-terminal (C, amino acids 501-671) domain of TCF4 were generated ( Figure 3A). The luciferase activities in the cells co-expressing GAL4-TWIST1 and full length (FL) and/or C-terminal (C) domain of VP16-TCF4 were higher than those in the cells expressing GAL4-TWIST1 alone ( Figure 3A). To screen small molecules that target TCF4-TWIST1 interaction, HTS was performed using the mammalian two-hybrid system and 501 natural product compounds from a chemical library. Approximately 12 small molecules inhibited TCF4-TWIST1 interaction ( Figure 3B). To exclude the possibility of downregulation of luciferase activities resulting from severe cell death, the cytotoxic activity of 12 natural compounds identified in Figure 3B was measured. As shown in Figure 3C, all compounds (5 µg/mL), except for emodin (5 µg/mL), exhibited potent cytotoxic activities. This indicated that emodin inhibits TCF4-TWIST1 interaction without exerting cytotoxic effects. Emodin dose-dependently mitigated the GAL4-TWIST1 and VP16-TCF4-FL-induced upregulation of luciferase activities ( Figure 3D). HEK293T cells were incubated for 24 h with each hit compound (5 μg/mL) as indicated. Cell viability was measured by crystal violet staining and assay. (D) Emodin is a potential small molecule targeting the interaction of TCF4 and TWIST1. HEK293T cells transfected with GAL4-TWIST1, VP16-TCF4-full length (FL) and pGL4.31-luciferase plasmids were incubated for 24 h with emodin as indicated, then luciferase activities were analyzed. The values represent the mean ± SD of three independent experiments performed in duplicate; * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis was performed by using a one-way ANOVA Tukey post hoc test.

Emodin Inhibits TCF4/TWIST1 Complex-Induced PTHLH Expression
Next, we examined whether emodin was sufficient to dissociate the TCF4/TWIST1 complex. Treatment with emodin decreased the interaction between TCF4 and TWIST1 ( Figure 4A). As shown in Figure 4B, emodin sufficiently inhibited the interaction between TCF4 and bHLH domain of TWIST1-NT2 (amino acids 1-165). These results indicate that emodin suppresses the interaction between TCF4 and TWIST1. As the TCF4/TWIST1 complex markedly upregulated PTHLH expression, the suppressive effect of emodin on TGFβ1-induced PTHLH and EMT-related gene expression was investigated. The expression of TGFβ1-induced PTHLH and EMT-promoting CDH2 mRNAs was downregulated in emodin (20 µM)-treated NCI-H358 ( Figure 4C) and A549 ( Figure 4D) cells. This indicated that emodin exerts potential therapeutic effects on cachexia by downregulating the expression of PTHLH and EMT-related genes.
TGFβ1-induced PTHLH and EMT-related gene expression was investigated. The expression of TGFβ1-induced PTHLH and EMT-promoting CDH2 mRNAs was downregulated in emodin (20 μM)-treated NCI-H358 ( Figure 4C) and A549 ( Figure 4D) cells. This indicated that emodin exerts potential therapeutic effects on cachexia by downregulating the expression of PTHLH and EMT-related genes. with TGFβ1 (20 ng/mL) for 1 h prior to emodin treatment, followed by further incubation with or without emodin (20 μM) for 24 h. CDH2 and PTHLH mRNA levels were measured by qRT-PCR. The values represent the mean ± SD of three independent experiments performed in duplicate; * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis was performed by using a one-way ANOVA Tukey post hoc test.

Emodin-Enriched Polygonum cuspidatum Extract (Pc-Ex) Inhibits TCF4-TWIST1 Interaction and Consequently Suppresses TGFβ1-Induced PTHLH Expression
Tumour-driven cytokines, such as growth differentiation factor 11, IL-1β, IL-6 leukaemiaia inhibitory factor, TNF-α, and TGFβ1 promote cancer cachexia with anorexia in the brain, proteolysis in the skeletal muscles, and browning in the WAT [5]. Thus, appetite-stimulating agents, such as megestrol (a synthetic derivative of naturally occurring progesterone) are widely used to treat cancer cachexia [41]. To develop a major component of medical nutrition therapy (MNT) for the treatment of lung cancer-induced cachexia, emodin-containing Pc-Ex was developed. The emodin content in Pc-Ex was confirmed using high-performance liquid chromatography (HPLC) ( Figure 5A). Pc-Ex dosedependently mitigated the GAL4-TWIST1 and VP16-TCF4-induced upregulation of luciferase activities ( Figure 5B). Additionally, Pc-Ex sufficiently suppressed the interaction between TCF4 and TWIST1 ( Figure 5C). Furthermore, Pc-Ex significantly mitigated the TGFβ1-induced upregulation of PTHLH mRNA expression in NCI-H358, A549, and Lewis Lung Carcinoma 1 (LLC1) cells ( Figure 5D). Pc-Ex downregulated the PTHrP levels in LLC1 cells ( Figure 5E). These results indicate that emodin-containing Pc-Ex is a potential agent for MNT-based treatment of cancer cachexia. Interaction of TCF4 and TWIST1 was measured by using co-immunoprecipitation assay and Western blotting. (C,D) Emodin suppresses TGFβ1-induced PTHLH expression in NCI-H358 and A549 lung cancer cells. (C) NCI-H358 and (D) A549 cells were incubated with TGFβ1 (20 ng/mL) for 1 h prior to emodin treatment, followed by further incubation with or without emodin (20 µM) for 24 h. CDH2 and PTHLH mRNA levels were measured by qRT-PCR. The values represent the mean ± SD of three independent experiments performed in duplicate; * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis was performed by using a one-way ANOVA Tukey post hoc test.

Emodin-Enriched Polygonum cuspidatum Extract (Pc-Ex) Inhibits TCF4-TWIST1 Interaction and Consequently Suppresses TGFβ1-Induced PTHLH Expression
Tumour-driven cytokines, such as growth differentiation factor 11, IL-1β, IL-6 leukaemiaia inhibitory factor, TNF-α, and TGFβ1 promote cancer cachexia with anorexia in the brain, proteolysis in the skeletal muscles, and browning in the WAT [5]. Thus, appetite-stimulating agents, such as megestrol (a synthetic derivative of naturally occurring progesterone) are widely used to treat cancer cachexia [41]. To develop a major component of medical nutrition therapy (MNT) for the treatment of lung cancer-induced cachexia, emodin-containing Pc-Ex was developed. The emodin content in Pc-Ex was confirmed using high-performance liquid chromatography (HPLC) ( Figure 5A). Pc-Ex dose-dependently mitigated the GAL4-TWIST1 and VP16-TCF4-induced upregulation of luciferase activities ( Figure 5B). Additionally, Pc-Ex sufficiently suppressed the interaction between TCF4 and TWIST1 ( Figure 5C). Furthermore, Pc-Ex significantly mitigated the TGFβ1-induced upregulation of PTHLH mRNA expression in NCI-H358, A549, and Lewis Lung Carcinoma 1 (LLC1) cells ( Figure 5D). Pc-Ex downregulated the PTHrP levels in LLC1 cells ( Figure 5E). These results indicate that emodin-containing Pc-Ex is a potential agent for MNT-based treatment of cancer cachexia.  attenuates TGFβ1-induced PTHLH expression in NCI-H358, A549, and LLC1 lung cancer cells. Cells were incubated with TGFβ1 (20 ng/mL) for 1 h prior to Pc-Ex treatment, followed by further incubation with or without Pc-Ex (50 and 100 µg/mL) for 24 h. PTHLH mRNA levels were measured by qRT-PCR. The values represent the mean ± SD of three independent experiments performed in duplicate; * p < 0.05, ** p < 0.01 and *** p < 0.001. Statistical analysis was performed by using a one-way ANOVA Tukey post hoc test. (E) Pc-Ex decreases PTHrP upon TGFβ1. LLC1 cells were incubated for 48 h in the absence or presence of 100 µg/mL of Pc-Ex with 20 ng/mL of TGFβ1. Mouse PTHrP protein levels were measured by using ELISA assay. The values represent the mean ± SD of three independent experiments performed in duplicate; ** p < 0.01. Statistical analysis was performed by using the unpaired Student's t-test.

Pc-Ex Attenuates Lung Cancer-Induced Cachexia
Next, the pharmacological effect of Pc-Ex on lung cancer-induced cachexia was examined. Non-tumour-bearing (NTB) and A549 tumour-bearing BALB/c-nu mice were fed with a standard animal diet (AIN93G) or a feed supplemented with 2% Pc-Ex when the volume of the tumours reached approximately 100 mm 3 . The bodyweight (carcass weight) of A549 tumour-bearing mice was lower than that of NTB mice ( Figure 6A). Additionally, the bodyweights of 2% Pc-Ex-administered A549 tumour-bearing mice were higher than those of the AIN93G-fed mice ( Figure 6A). This indicated that emodin-containing Pc-Ex exerts therapeutic effects on body weight loss in tumour-bearing mice. Previous studies have reported that emodin exerts anti-cancer effects [42][43][44]. Consistently, the findings of this study indicated that the tumour growth in 2% Pc-Ex-administered mice was lower than that in the AIN93G-fed mice ( Figure 6B). Additionally, A549 tumour-induced gastrocnemius muscle atrophy in 2% Pc-Ex-fed mice was alleviated when compared with that in AIN93Gfed A549 tumour-bearing mice ( Figure 6C). Tumour-induced loss of gastrocnemius muscle weight was mitigated upon treatment with 2% Pc-Ex ( Figure 6D). The PTHLH mRNA and PTHrP protein levels were downregulated in A549 tumours from 2% Pc-Ex-fed mice ( Figure 6E,F). Next, the effect of emodin-containing Pc-Ex on the upregulated expression of white adipose (WAT) metabolism and skeletal muscle atrophy-related genes in tumourbearing mice was examined. As shown in Figure 6G, the A549 tumour-induced expression of skeletal muscle atrophy-related genes, such as Mstn, Fbxo32, and Trim63 was mitigated upon treatment with 2% Pc-Ex. Similarly, the administration of 2% Pc-Ex mitigated the A549 tumour-induced upregulation of WAT metabolism-related gene (Pgc1a and Acox1) in epididymal WAT ( Figure 6H). These results indicate that emodin-containing Pc-Ex can mitigate the wasting of fat and skeletal muscle in lung cancer-induced cachectic mice.

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Discussion
PTHrP, an essential paracrine and autocrine ligand encoded by PTHLH, regulates bone homeostasis and the initiation, growth, and progression of various types of cancer [14]. The upregulated expression of oncogenic PTHLH is associated with poor prognosis owing to its role in promoting tumour initiation, growth, angiogenesis, metastasis, and chemoresistance in pancreatic [19], colorectal [18], intrahepatic cholangiocarcinoma [45], head and neck [46], osteosarcoma [47], and breast cancers [17,48]. In this study, the correlation between PTHLH expression and LUAD and LUSC was examined using bioinformatics analysis. The analysis of TCGA datasets revealed that the PTHLH mRNA levels in the LUAD and LUSC tissues were upregulated when compared with those in the nontumorous lung tissues. Additionally, the upregulated PTHLH mRNA levels were correlated with decreased survival in patients with LUAD. These results indicate that PTHLH is a potential diagnostic biomarker and a molecular therapeutic target for lung cancer.
Lung cancer-derived PTHrP promotes the wasting of WAT and skeletal muscles, which results in cancer cachexia [4]. However, the precise molecular mechanisms under-

Discussion
PTHrP, an essential paracrine and autocrine ligand encoded by PTHLH, regulates bone homeostasis and the initiation, growth, and progression of various types of cancer [14]. The upregulated expression of oncogenic PTHLH is associated with poor prognosis owing to its role in promoting tumour initiation, growth, angiogenesis, metastasis, and chemoresistance in pancreatic [19], colorectal [18], intrahepatic cholangiocarcinoma [45], head and neck [46], osteosarcoma [47], and breast cancers [17,48]. In this study, the correlation between PTHLH expression and LUAD and LUSC was examined using bioinformatics analysis. The analysis of TCGA datasets revealed that the PTHLH mRNA levels in the LUAD and LUSC tissues were upregulated when compared with those in the non-tumorous lung tissues. Additionally, the upregulated PTHLH mRNA levels were correlated with decreased survival in patients with LUAD. These results indicate that PTHLH is a potential diagnostic biomarker and a molecular therapeutic target for lung cancer.
Lung cancer-derived PTHrP promotes the wasting of WAT and skeletal muscles, which results in cancer cachexia [4]. However, the precise molecular mechanisms underlying the production and secretion of PTHrP in lung cancer cells are poorly understood. TGFβ1, a well-known secreted protein from the tumour, immune, and tumour-associated fibroblast cells, promotes tumour cell migration, invasion, and metastasis [49]. Additionally, TGFβ1 functions as a critical stimulus for promoting cancer cachexia, which is characterised by severe bodyweight loss resulting from skeletal muscle loss and WAT browning [7]. Based on these findings, we hypothesized that TGFβ1-primed tumour cells exhibit PTHrP expression in lung cancer cells. Consistent with the findings of previous studies, this study demonstrated that the expression of PTHLH was upregulated in response to TGFβ1 signalling in various lung cancer cell lines [38][39][40]. This suggests that PTHLH is a critical component for TGFβ1-induced cachexia in lung cancer.
TCF4 (also known as ITF2 and E2-2), a class I bHLH transcription factor, functions as a transcriptional activator or a transcriptional repressor depending on its binding partners [24]. Previous studies have reported that TCF4 regulates cancer development and progression [25,26,29,50,51]. Additionally, TCF4 is reported to regulate the transcription of pro-metastatic and EMT-related genes in malignant melanoma and lung cancer [24,26]. TGFβ1 signalling upregulates TCF4 expression in cancer cells [16,52]. However, the clinical roles of TCF4 in cancer cachexia have not been previously investigated. Thus, we hypothesized that TCF4 could function as an upstream transcription factor for TGFβ1 signalling-induced PTHLH expression. The PTHLH mRNA levels were upregulated upon TCF4 overexpression and downregulated upon TCF4 silencing in lung cancer cells. These results indicate that TCF4 is an essential transcription factor that regulates TGFβ1-mediated PTHLH expression.
TCF4 can interact with multiple types of transcription factor and function as a transcriptional activator or a transcriptional repressor depending on its binding partners during various biological processes, such as cell cycle, proliferation, differentiation, and development [17]. The interaction between Tcf4 and Math1 regulates neuronal progenitor differentiation and is associated with the pathogenesis of mental disorders [53]. TWIST1, a bHLH transcription factor, plays an essential role during embryonic development through the regulation of EMT-related genes [31]. Previous studies have demonstrated that TCF4 can interact with TWIST1 to promote the expression of EMT-related genes, such as CDH2, VIM, SNAI1, and SNAI2, in embryonic stem and lung cancer cells [16,32]. This study demonstrated that TCF4 strongly binds to the bHLH domain of TWIST1 to form a heterodimer. The interaction between TCF4 and TWIST1 was confirmed using the mammalian two-hybrid interaction assay. TCF4-TWIST1 interaction regulates TGFβ1 signalling-induced EMT and cancer metastasis [14]. However, the functional role of the TCF4/TWIST1 complex in PTHrP-associated cancer cachexia has not been previously investigated. In this study, the PTHLH mRNA levels in A549 and NCI-H358 cells co-expressing TCF4 and TWIST1 were higher than those in cells expressing TCF4 or TWIST1 alone. These results suggest that TCF4/TWIST1 complex-induced PTHLH expression may promote cachexia in lung cancer and that these molecular frameworks are promising therapeutic targets for cancer cachexia.
Polygonum cuspidatum (also known as Reynoutria japonica) is a traditional Chinese medicinal herb with a wide range of pharmacological activities, including anti-inflammatory, anti-cancer, and anti-hyperlipidaemic activities [54]. Various active compounds, including emodin (quinone), resveratrol (stilbene), and quercetin (flavonoid), have been isolated from the P. cuspidatum. These phytochemicals are reported to exert beneficial effects on various chronic diseases [54]. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) exerts multiple pharmacological effects, including anti-allergic, anti-osteoporotic, anti-diabetic, neuroprotective, hepatoprotective, cardioprotective, and anti-cancer effects [55,56], on various chronic diseases. A pre-clinical pharmacokinetic study has demonstrated the safety of emodin in mice [57]. Treatment with low (20 mg/kg body weight), medium (40 mg/kg body weight), or high (80 mg/kg body weight) doses of emodin for 12 weeks did not affect the physiology of major organs, such as the kidneys and liver. This indicated the potential application of emodin, a natural ingredient, for the treatment of human chronic diseases [57]. Emodin is reported to suppress pancreatic cancer-induced cachexia [58]. Additionally, emodin inhibited tumour-induced hepatic gluconeogenesis and skeletal muscle proteolysis in MiaPaca2 cell-implanted athymic mice [58]. Although emodin is reported to inhibit TGFβ1 signalling [59][60][61][62], the suppressive effects of emodin on TGFβ1associated cancer cachexia have not been previously investigated. In this study, Pc-Ex, which contains emodin as a major component, sufficiently suppressed the PTHLH mRNA and PTHrP expression levels in lung cancer cells stimulated with TGFβ1 by inhibiting the interaction between TCF4 and TWIST1. Additionally, the administration of Pc-Ex downregulated PTHLH expression in A549 tumour-bearing mice and significantly decreased tumour growth, increased skeletal muscle weight, reversed the expression of proteolysis and fat browning-related genes in the skeletal muscle and WAT, respectively, in cachectic mice. These findings demonstrate that emodin-enriched Pc-Ex is a potential therapeutic for cancer cachexia.
Mice were fed on an AIN93G diet supplemented with 2% Pc-Ex in the form of edible pellets for a month. The daily treatment dose of Pc-Ex was 2400 mg/kg body weight for each mouse. HPLC analysis of emodin contents in Pc-Ex indicated that the dietary emodin treatment dose was 12 mg/kg body weight. Previous studies have revealed the hepatotoxic effects of emodin [57]. Although Pc-Ex-formulated diets comprise low amounts of emodin, mouse hepatic metabolomic profile must be examined for the application of Pc-Ex to suppress tumour growth and cachexia. The contents and half-life of emodin in the blood of mice administered with Pc-Ex can be analysed using pharmacokinetic analysis, which can aid in determining the pharmacological potential of emodin for cancer-induced cachexia.
Mechanistically, the expression of PTHLH in multiple cancers is regulated by various transcription factors and signalling cascades. The inactivation or downregulation of p38MAPK signalling is reported to suppress PTHLH expression in lung and liver metastases of primary colorectal cancer [63]. Smad3, a transcription factor, upregulates the transcription of PTHLH by binding to the proximal PTHLH promoter region in response to TGFβ1 signalling in breast cancer cells and consequently promotes bone metastasis [17]. The Runx2-mediated activation of PTHLH expression promotes the growth of head and neck squamous cell carcinoma [46]. In this study, the interaction between TCF4 and TWIST1 upregulated PTHLH expression and consequently promoted lung cancer growth and cachexia. These findings revealed the mechanistic framework involved in the regulation of PTHLH expression, which can aid in the development of novel therapeutic strategies for lung cancer metastasis and lung cancer-induced cachexia.

Conclusions
The interaction between TCF4 and TWIST1 upregulated PTHLH expression in lung cancer cells in response to TGFβ1 stimulation. This indicated that this transcriptional framework may be involved in the pathogenesis of lung cancer-induced cachexia. The major findings of this study are as follows: TCF4 promoted TWIST1-mediated PTHLH expression; emodin and Pc-Ex suppressed TGFβ1-induced PTHLH expression by inhibiting the interaction between TCF4 and TWIST1; emodin-containing Pc-Ex alleviated skeletal muscle wasting and fat browning and downregulated the expression of atrophy-associated genes in tumour-bearing mice. Thus, the findings of this study indicated that the interaction between TCF4 and TWIST1 is a potential molecular therapeutic target for lung cancer-induced cachexia and that emodin and emodin-enriched Pc-Ex, which inhibit TCF4-TWIST1 interaction, are promising therapeutic candidates for lung cancer-induced cachexia.

Institutional Review Board Statement:
The animal experiments were approved and performed in accordance with the guidelines of Konkuk University Institutional Animal Care and Committee (KU20062).

Informed Consent Statement: Not applicable.
Data Availability Statement: All materials generated in this study are available from the corresponding author on reasonable request.