KIT-c-Jun Signaling Up-regulated HMGCS1 to Promote Colorectal Cancer Development

doi:10.21203/rs.3.rs-1508182/v2

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Research Article

KIT-c-Jun Signaling Up-regulated HMGCS1 to Promote Colorectal Cancer Development

https://doi.org/10.21203/rs.3.rs-1508182/v2

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Purpose

Colorectal cancer (CRC) ranks 2nd in morbidity and 3rd in mortality among all cancers worldwide. 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) is ubiquitously expressed in humans. Remarkably, the mevalonate pathway is often dysregulated in many cancers, suggesting tumor dependency on this classic metabolic pathway. In addition to altered substrates in the mevalonate pathway, it was recently found that HMGCS1 was highly expressed in prostate, melanoma and breast cancers. The transmembrane glycoprotein KIT is critical for the survival and development of various cells through activating downstream pathways and targets. Our previous research has proved that KIT is hyper-expressed in CRC and promotes CRC progression. However, whether HMGCS1 mediates the tumor-promoting effect of KIT signaling has not been unclosed.

Material and Methods

We investigated the regulatory mechanism of KIT signaling on HMGCS1 expression and the role of increased HMGCS1 in CRC development by the use of gene over-expression and knock-down techniques in CRC cells, biological function tests, protein-DNA binding detection, bioinformatics, database analysis and c-kit loss-of-function mutant mice (Wads^m/m).

Results

HMGCS1 was up-regulated by KIT-JNK-c-Jun signaling in CRC cells and could be a biomarker for CRC. Highly expressed HMCGS1 promoted CRC cell proliferation and invasion while inhibiting apoptosis. Our results provided evidence for the role of HMGCS1 in CRC progression and suggested that blocking KIT-JNK-c-Jun-HMGCS1 might be a new strategy for treating CRC patients.

Conclusion

Our results indicated that KIT signaling could up-regulate HMGCS1 in CRC mediated by Ap-1/c-Jun. Hyper-expressed HMGCS1 promotes CRC cell proliferation and invasion while inhibiting apoptosis.

HMGCS1

Ap-1/c-Jun

KIT

Colorectal Cancer

Proliferation

Invasion

Apoptosis

Cancer is one of the leading causes of death and one of the biggest challenges to increasing life expectancy. It is also one of the costliest diseases, for more than 133 billion dollars was spent on treatment and supportive care worldwide in 2017. Colorectal cancer (CRC) ranks 2nd in morbidity and 3rd in mortality among all cancers worldwide. According to estimates from GLOBOCAN, over 1.8 million new CRC cases and 881,000 deaths are estimated to occur in 2018 [1]. CRC incidence and mortality are rapidly growing worldwide, especially in China. With the high-speed socio-economic development, CRC incidence and mortality in China have been above the global average, being a severe threat to people's health and a heavy burden on medical care. Proctocolectomy and adjuvant chemotherapy, radiotherapy, targeted therapy, and supportive care are currently the main treatments for CRC patients. Unfortunately, the CRC patient's overall survival has not improved significantly due to frequent relapse and metastasis, closely related to oncogenes aberrant expression and activation. Therefore, it is essential to explore the critical molecules in CRC development, which helps research and develop effective and precise targeted therapeutic drugs.

Despite serial breakthroughs in CRC studies, the molecular mechanism underlying the tumorigenesis and progress of CRC remains largely unknown. For the last decades, cholesterol metabolism in cancers has attracted many researchers. Cholesterol is vital for the survival and growth of mammalian cells, which is synthesized mainly through the mevalonate pathway. More than a membrane constituent, cholesterol can initiate or promote cancers by being a precursor to bile acids and steroid hormones, modulating signaling pathways involved in tumorigenesis and progression, and facilitating the formation of specialized membrane microdomains [2]. Cholesterol level is intensively correlated with CRC incidence [3]. Red meat consumption, a significant source of dietary cholesterol, is suggested as causative in CRC, possibly explaining the increasing CRC. It is well-documented that the mevalonate pathway is often dysregulated in many cancers, guiding tumor dependency on this classic metabolic pathway [4]. In addition to the altered substrates in the mevalonate pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) was the rate-limiting enzyme within the mevalonate pathway was over-expressed, contributing to the oncogenic progression and poor prognosis [5, 6]. Besides HMGCR, 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1) plays a vital role in cholesterol biosynthesis. Also, HMGCS1 was highly expressed in prostate, melanoma, and breast cancers [7, 8]. However, research on the expression, regulation, and role of HMGCS1 in CRC is lacking. One study on the transcriptome network approach suggested that HMGCS1 was up-regulated in CRC samples. It could be a novel testable target to eliminate CRC cells directly or potentially through lipid-lowering drugs associated with selected anticancer drugs [9].

The transmembrane glycoprotein KIT (also known as c-KIT or CD117) is a member of the sub-family of receptor tyrosine kinases (RTKs), which is essential for the survival and development of various cells, including gastrointestinal pacemaker cells (interstitial cells of Cajal, ICC), mast cells, melanocytes, germ cells and erythrocytes [10] KIT is activated by binding to its ligand stem cell factor (SCF), resulting in various downstream pathways such as MAPK, SRC, JAK/STAT and PI3K/AKT that promote cell proliferation, survival and inhibit apoptosis [11]. Yet, over-expressed or over-activated KIT/SCF signaling is implicated in the onset and progression of multiple cancers, including sinonasal lymphoma, seminomas, systemic mastocytosis, melanomas, acute myeloid leukemia, and gastrointestinal stromal tumors [12, 13]. The role of KIT/SCF signaling has also been evaluated in CRC. Up to 51% of CRC patients are KIT^high, and the hyper-activated KIT/SCF signaling promotes the growth of colon xenograft tumors [14, 15]. Consistently, our previous research showed that c-kit loss-of-function mutant mice (Wads^m/m) had much fewer and smaller colon tumors induced by AOM + DSS treatment than wild-type (WT) mice did. Activating KIT/SCF signaling in vitro accelerated CRC cell proliferation and invasion, reversed by RTK inhibitor Imatinib [16]. Therefore, we wonder whether HMGCS1 mediates the tumor-promoting effect of KIT/SCF signaling in CRC.

Bioinformatics analysis

The University of California, Santa Cruz (UCSC) database was used to find the sites 2000 bp upstream and 100 bp downstream of Ap-1 TSS [17]. PROMO program has projected possible TFs capable of binding to the HMGCS1 promoter [18,19]. The cBioPortal results from The Cancer Genome Atlas (TCGA) have been used to analyze HMGCS1 and AP-1 expression levels in patients with or without recurrences of CRC [20,21]. The GEPIA2 server was used to study mRNA expression and the association between the two genes in HMGCS1 and Ap-1 in CRC and normal tissues [22].

Cell culture & treatment

The CRC cell lines used in the experiment include HCT-116, Caco-2, RKO, LAS174T, HT-29, and HEK293T were grown in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100mU/ml of Penicillin (Gibco) and 100µg/ml of Streptomycin (Gibco) at 37ºC in a Humidified incubator of 5% CO₂. The medium was changed every 24hr. All cell lines were purchased from American Tissue Culture (ATCC).

Reagents included 50 ng/ml recombinant human stem cell factor (rhSCF, 255-SC, R&D Systems, USA) added for 15 minutes, 2 µmol/L Imatinib (STI571, S1026, Selleck, Shanghai, China) added for 2.5 hr and 30 µmol/L ISCK03 (ab145066, Abcam) added for 2.5 hr.

Over-expression of AP-1 by plasmid transfection

AP-1 over-expression and its negative control plasmid were constructed by GeneChem Technology Co., Ltd (Shanghai, China). We co-transfected the reporter plasmids with AP-1 overexpressing plasmid into Caco-2 cells using Lipofectamine ^TM 2000 (Thermo Scientific). The vector scheme is shown in Figure S1.

Over-expression of KIT by lentivirus infection

Lentivirus used for KIT stable over-expression was constructed by Shanghai GeneChem Technology Co., Ltd (Shanghai, China) and transfected into CRC cells using Lipofectamine ^TM 2000 (Thermo Scientific).

Knock-down of HMGCS1 by siRNA

Both siRNA siHMGCS1 and siNC were purchased from The RiboBio (Guangzhou, Guangdong, China). According to the manufacturer's protocol, all the transfection was performed using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Inc.). Cells were cultured into a 60mm dish when the cells were in 80% to 90% confluency. Then, the complete DMEM into antibiotic and FBS-free DMEM for 24 hours. To prepare Lipofectamine reagent, take 250µl of Opti-MEM and 10µl Lipofectamine reagent per well and put them for 5 minutes at room temperature. For the preparation of siRNA solution taken, 250µl of Opti-MEM and 2500-5000ng siRNA per well. Then gently mix Lipofectamine reagent and DNA at room temperature for 20 minutes. After this, discarded DMEM and added Lipofectamine reagent DNA mixture into the dish. Add 1.5ml of Opti-MEM to make the volume up to 2ml per well. After 24/48 hrs., check the inflorescence intensity to evaluate transfection efficiency. If transfection was successful, then harvest cells. The vector scheme is shown in Figure S2.

Chromatin immunoprecipitation (ChIP)

ChIP assay was performed according to the manufacturer's instructions (EZ-Magna ChIP^TM A Kit, #17-408, Merck-Millipore, USA). Chromatin was immunoprecipitated for 24hr at 4 ̊C using rabbit monoclonal anti-Ap1. Primers are shown in Table 1.

Table 1. Primers sequences

Genes	qRT-PCR Primer Sequences
KIT	Forward: 5’-CAGGCAACGTTGACTATCAGT-3’ Reverse: 5’-ATTCTCAGACTTGGGATAATC-3’
HMGCS1	Forward: 5’-GCTCTTGGGATGGACGGTAT-3’ Reverse: 5’-GCGGTCTAATGCACTGAGGT-3’
c-Jun	Forward: 5’-TATGAAGTGAGTCATGGGCAA-3’ Reverse: 5’-TATGAAGCAGACACTGGGCAA-3’
GAPDH	Forward: 5’- CCTGCACCACCAACTGCTTA -3’ Reverse: 5’- AGTGATGGCATGGACTGTGG -3’
ChIP- qPCR primer sequences
Ap1 promoter	Forward:5’-TATGAAGTGAGTCATGGGCAA-3’ Reverse: 5’-TATGAAGCAGACACTGGGCAA-3’

Real-time cellular analyzer (RTCA)

RTCA (ACEA Biosciences, USA) was used to monitor cell proliferation, and invasion as previously described [23].

Cell apoptosis detection by flow cytometry (FCM)

The cells were fixed with 70% ethanol in PBS and routinely kept at -20C overnight. The cells were washed with PBS and permeabilized with PBS, 4% fetal bovine serum, and 0.1% Triton X-100 for 10 min ice. Then cells were incubated within 1:200 dilutions in PBS, 4% fetal bovine serum for 2hr. Cells were washed twice with PBS-T and incubated with 1:200 dilutions of fluorescein-tagged goat anti-mouse secondary antibody. After washing with PBS-T, cells will be suspended in PBS and analyzed using a flow cytometer.

Western blot

A mixture of Pierce RAPI Lysate (Applygen, Beijing, China) and Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Inc.) was used to lyse cells at a ratio of 100:1. The protein concentration was measured using the BCA method using NanoDrop 2000c (Thermo Scientific, Gene Company Limited). The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked with 5% bovine serum albumin (BSA) and incubated with primary antibodies against HMGCS1 (1:1000), p-c-JUN (1:1000), C-JUN (1:1000), C-KIT (1:1000), P-KIT (1:1000), and GAPDH (1:5000) for overnight at 4ºC. Then membranes were incubated with goat anti-rabbit secondary antibody (1:5000) for 2 hours at 37ºC. Super Signal West Pico Chemiluminescent substrate (Thermo Fisher Scientific, Inc.) visualized the protein bands. ImageJ 1.50i (National Institutes of Health, Bethesda, MD, USA) was used to quantify the protein bands.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from cell lines or tissues using TRIzol (Invitrogen, Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the 5x All In One RT Master Mix (ABM, Canada). The expression was measured using the SYBR Green Script miRNA RT-PCR kit (Takara) with the AB1 7500 Real-time PCR system (Bio-Rad Laboratories Inc., Hercules, CA, USA). All of these steps were operated according to the manufacturer's protocol. The oligonucleotide sequences of the qRT-PCR primers are listed in Table 1.

Cell proliferation measured by CCK8 cell viability assay

Following the manufacturer's protocol, cell proliferation was measured using the CCK8 assay Kit (Dojindo Laboratories, Japan). The cell was plated in 96-well plates and treated with drugs. After 72 hrs. Incubation of 10µl CCK8 was added to each well for an additional 2 hrs. Then absorbance was measured by a Multiskan FC photometer at 450nm.

c-Kit loss of functional mutant mice (Wads^m/m)

Five c-kit loss of functional mice (Wads^m/m) and %WT littermates were used on the C57BL/6 background and used in a previous study [24]. Animal studies were performed under strict protocols approved by the Animal Care and Use Committee of Capital Medical University.

Tissue specimen and clinicopathological information

Specimen of tumor and adjacent normal tissues of the colon were obtained from 30 patients who had been pathologically diagnosed with colorectal cancer and underwent surgical resection. Tissue specimens were ground then sonicated in the lysis buffer (50 mM Tris-HCL, pH 7,4, 1mM EGTA, 150 mM Nack, 5% Triton X-100) with protease inhibitors. The samples were micro centrifuged to remove the large debris and subjected to western analysis. All patient-derived specimens were collected from Beijing Friendship Hospital, Capital Medical University Beijing, and archived under complete protocols approved by the Research Board of Capital Medical University.

Statistical analysis

The student's t-test (two-tailed) was performed for comparisons between groups in cell proliferation assay and gene expression analysis by GraphPad Prism 5. Significance was presented as a p-value <0.005 (*), <0.01 (**) and <0.001 (***), non-significant differences were presented as NS.

HMGCS1 was hyper-expressed in CRC tissues

HMGCS1 expression was detected by qPCR and Western blot in the 30 pairs of CRC tissues and corresponding para-tumoral mucosa. The results showed that HMGCS1 was overexpressed in tumors compared with that in para-tumoral normal mucosa (Fig. 1a and b), which was consistent with the analysis on the TCGA database (Fig. 1c). These results suggested a potential role of aberrant HMGCS1 in CRC development.

AP-1/c-Jun was a candidate for HMGCS1 transcription

Protein expression is controlled by a distinct regulatory network, among which transcriptional regulation is one of the fundamental and general mechanisms. Predicted transcription factors (TFs) that can bind to the HMGCS1 promoter using bioinformatics analysis. The maximum matrix dissimilarity is zero, with 18 output TFs (Fig. 2a). In this study, we had a particular interest in the transcription factor (TF) AP-1/c-Jun due to the reasons: ① AP-1/c-Jun was able to bind to the HMGCS1 promoter indicated by UCSC, and ALLGEN-PROMO bioinformatics (Fig. 2b and c). ② AP-1/c-Jun was highly expressed and activated in CRC compared with para-tumoral mucosa (Fig. 2d).

AP-1/c-Jun accelerated HMGCS1 transcription by binding to its promoter

To confirm the regulatory role of AP-1/c-Jun on HMGCS1 transcription, we analyzed the mRNA (FPKM value) level of AP-1/c-Jun and HMGCS1 in 275 CRC patients from the TCGA database, which revealed a significant positive correlation between them (Fig. 3a). We performed ChIP to determine whether the AP-1/c-Jun can bind to the predicted site within the HMGCS1 promoter. After activating AP-1/c-Jun by 12-O-Tetradecanoylphorbol 13-acetate (TPA), a PKC activator can also activate AP-1, the binding of Ap-1/c-Jun to the HMGCS1 promoter was significantly increased compared with the IgG group (Fig. 3b and c). Moreover, we evaluated the effect of Ap-1/c-Jun in HMGCS1 expression. Ap-1/c-Jun was over-expressed in Caco-2 cells, which in addition to that, had significantly up-regulated HMGCS1 (Fig. 3d).

KIT-c-Jun signaling pathway up-regulated HMGCS1

We previously reported that KIT was highly-expressed in CRC (Fig. 4a and b), and AP-1/c-Jun was regulated by KIT signaling via the JNK pathway in CRC cells (Fig. 4c) [25]. To determine the effect of KIT signaling on HMGCS1 expression, KIT signaling was over-activated by lentivirus mediation or blocked by its inhibitor Imatinib, an RTK inhibitor, or ISCK03, a specific KIT/SCF signaling inhibitor, in HCT-116 cells. Figures 4d and 2 show that KIT over-activated cells had highly activated AP-1/c-Jun and HMGCS1. While the activity of AP-1/c-Jun was inhibited, the expression of HMGCS1 was reduced in cells treated with either Imatinib or ISCK03. In addition, we compared expressions of AP-1/c-Jun and HMGCS1 in the c-kit loss-of-function mutant Wads^m/m mice and WT littermates in vivo. The results showed that along with the reduced KIT activity, the expression of AP-1/c-Jun and HMGCS1 significantly decreased (Fig. 4f). These results confirmed that in CRC cells, the hyper-activated KIT signaling elevated HMGCS1 expression via activating AP-1/c-Jun.

Knock-down of HMGCS1 reduced CRC cell proliferation and invasion

Finally, we evaluated the role of HMGCS1 in the biological functions of CRC cells. HMGCS1 was knocked down by introducing its specific siRNA (Fig. 5a and b). The cell proliferation was significantly inhibited in CRC cells as measured by CCK8 assays and a real-time cellular analyzer (RTCA) (Fig. 5c and 5). Likewise, the invasiveness hindered HMGCS1 knocked-down cells (Fig. 5e and 5).

Knock-down of HMGCS1 increased apoptosis of CRC cells

The CRC cell apoptosis was detected by flow cytometry. The knock-down of HMGCS1 resulted in a marked increase in cell apoptosis (Fig. 6).

In this study, we, for the first time, reveal that the KIT-JNK-c-Jun signaling pathway up-regulates the HMGCS1 in CRC cells, which promotes CRC progression.

Reprogramming of various metabolic pathways has been implicated in the multistep development of tumors. The first discovered metabolic reprogramming is the shift from catabolic to anabolic metabolism, known as the Warburg effect, which is considered a classic hallmark of cancer cells [26]. As early as a century ago, cholesterol accumulated in malignant tissues [27], spotlighting the cholesterol metabolic reprogramming in cancer biology. Reprogrammed cholesterol metabolism is also recognized as a hallmark feature in cancer cells. Many cancer cells exhibit dysregulation of the mevalonate pathway, the fundamental way for cholesterol biosynthesis [28–30]. A clear association between CRC and cholesterol accumulation prompted researchers to investigate the mevalonate pathway genes. For example, increased HMGCR, the rate-limiting enzyme in the mevalonate pathway, was frequently found in various cancer, including CRC [5, 6]. This study focused on HMGCS1, the immediately upstream enzyme of HMGCR in the mevalonate pathway, condensing acetyl-CoA and acetoacetyl-CoA to HMG-CoA [31]. The Association of HMGCS1 with other cancers has been described [7, 30, 32]; neither the role nor the regulation of HMGCS1 has not been wholly illuminated in CRC. Like HMGCR, there was a significant increase of HMGCS1 in CRC samples suggesting a possible tumor-promoting role of HMGCS1 in CRC progression. We verified that HMGCS1 could promote CRC cell proliferation and invasion using knock-down gene technology while inhibiting apoptosis. Our results were consistent with a new publication demonstrating that HMGCS1 could enhance cell proliferation, migration, and invasion of CRC cells [33].

The next question was how the expression of HMGCS1 was up-regulated in CRC. As bioinformatics technology develops rapidly, screening out the TFs that can bind to their potential target genes and control their transcription becomes easier. Here, we predicted that AP-1/c-Jun was one of the candidates for HMGCS1 transcription using UCSC and PROMO. ChIP assays further confirmed the prediction. The AP-1 consists of principal homodimers of the Jun family members with the Fos family members (c-Jun, Jun-B, Jun-D, c-Fos, Fos-B, Fra-1, and Fra-2) [34]. AP-1 activity is regulated by its dimer composition, determined by the differential expression of Jun and Fos families through the sequence of AP-1 binding sites. AP-1 has gotten attention due to its essential role in the basic biological process and cellular feedback to stimuli that regulate apoptosis, proliferation, oncogenic factor, and differentiation [35]. AP-1 controls both basal and inducible transcription of several genes containing AP-1 binding sites. AP-1 activity in the cells is regulated by a broad range of physiological and pathological stimulation along with oncogenic stimuli growth factors, cytokines, infection, and stress signals. Among all of these members, c-Jun is the fundamental component of the AP-1 complex and becomes transcriptionally active upon phosphorylation at Ser63 and Ser73 within its N-terminal transactivation domain by JNK [36]. C-Jun is frequently overexpressed in human cancers. Increased c-Jun transcriptional activity and medicated gene expression are related to ras-transformation [37].

The activity of AP-1/c-Jun was under the control of KIT signaling, as we previously demonstrated. KIT is a key member of RTKs, responsible for multiple cellular functions. More importantly, aberrant KIT has been found in many tumors. Over-expression or hyper-activated KIT signaling promotes cancer progression, including CRC. KIT elicits the tumor-promoting effect by mediating its targets via downstream pathways. In this study, we suggested that HMGCS1 was a new target of the KIT-JNK-c-Jun signaling pathway and the hyper-activated pathway was able to accelerate CRC progression.

Statins, the HMGCR inhibitors, have been prescribed for the treatment of various tumors in addition to cardiovascular diseases for their cholesterol-lowering property. However, recent clinical studies showed different efficacies of low-dose or high-dose statins combined with chemotherapeutics or targeted drugs [38–41]. HMGCR or HMGCS1 expression was even elevated post-atorvastatin/simvastatin treatment due to a restorative feedback response [42]. We hypothesized that blocking the feedback response to statins, for instance, inhibiting KIT-c-Jun, could potentiate anticancer efficacy.

We concluded that HMGCS1 was up-regulated by KIT-JNK-c-Jun signaling in CRC cells and could be a biomarker for CRC. Highly expressed HMCGS1 promoted CRC cell proliferation and invasion while inhibiting apoptosis. Our results provided evidence for the role of HMGCS1 in CRC progression and suggested that blocking KIT-JNK-c-Jun-HMGCS1 might be a new strategy for treating CRC patients.

In conclusion, our results indicated that KIT signaling could up-regulate HMGCS1 in CRC mediated by Ap-1/c-Jun. Hyper-expressed HMGCS1 promotes CRC cell proliferation and invasion while inhibiting apoptosis. Our research provided an experimental reference for developing KIT-Ap-1/c-Jun-HMGCS1 targeted therapeutics to treat CRC patients.

Author Contributions

Conceptualization, S.Y., and D.Z., Methodology, M.K.A., L.Y., H.S., T.S., B.W., J.M., X.L., H.C. and S.Y., Validation, S.Y. and D.Z., Formal Analysis, M.K.A., L.Y., and S.Y., Writing-Original Draft Preparation, M.K.A., Writing-Review & Editing, S.Y., and D.Z., Supervision, S.Y., and D.Z., Project Administration, S.Y. and D.Z., Funding Acquisition, T.S., B.W., S.Y., and D.Z.

Funding

This study was supported by the Beijing Natural Science Foundation (5222003, 5222004, 7172021, and 5202007), the National Natural Science Foundation of China (32071180 and 31801011), and the Support Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Five-year plan (IDHT20170516).

Ethics Approval:

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Capital Medical University Ethical Committee.

Conflict of Interests:

The authors declare no conflicts of interest.

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No competing interests reported.

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KIT-c-Jun Signaling Up-regulated HMGCS1 to Promote Colorectal Cancer Development

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