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Abstract

Lung cancers are broadly classified into small cell lung cancer and non–small cell lung cancer, with non–small cell lung cancer one of the leading causes of cancer-associated deaths worldwide. Presently, the mechanisms underlying lung tumorigenesis remain incompletely understood. Accumulating evidence indicates that abnormal expression of long non-coding RNAs is associated with tumorigenesis in multiple cancers, including lung cancer. HAGLR messenger RNA of non–small cell lung cancer tissues was significantly higher. Moreover, high levels of HAGLR expression were associated with non–small cell lung cancer tumor lymph node metastasis status, stage, and poor overall survival. Inhibition of HAGLR in non–small cell lung cancer cells suppressed cell proliferation and invasion. RNA interference–mediated downregulation of HAGLR also decreased levels of fatty acid synthase, with fatty acid synthase levels positively correlated with HAGLR expression in non–small cell lung cancer specimens. In addition, the cellular free fatty acid content of cancer cells was decreased following HAGLR knockdown. HAGLR depletion significantly inhibited the growth of non–small cell lung cancer cells in vivo. Furthermore, the expression levels of p21 and matrix metallopeptidase-9 (MMP-9) were dysregulated when HAGLR expression was suppressed. Our results suggest that HAGLR is an important regulator of non–small cell lung cancer cell proliferation and invasion, perhaps by regulating fatty acid synthase. Therefore, targeting HAGLR may be a possible therapeutic strategy for non–small cell lung cancer.

Keywords

Non–small cell lung cancer HAGLR fatty acid synthase de novo lipogenesis cell cycle invasion

Introduction

Non–small cell lung cancer (NSCLC) is the most common form of lung cancer, accounting for approximately 85% of all known lung cancer cases.¹ Furthermore, NSCLC is commonly locally advanced or metastatic at diagnosis. NSCLC can be further classified into one of the three subtypes—lung adenocarcinoma (LAD), large cell carcinoma, or lung squamous cell carcinoma (LSCC)—based on pathological characteristics. LAD and LSCC are the predominant subtypes, constituting approximately 50% and 40% of NSCLC cases, respectively.^2,3 Traditional therapeutic agents such as tyrosine kinase inhibitors that target the epidermal growth factor receptor,^4,5 anaplastic lymphoma kinase (ALK) inhibitors,⁶ and immune checkpoint inhibitors have been improved upon and are successfully used in clinical practice.⁷ However, the 5-year overall survival (OS) rate for lung cancer patients of all stages combined remains as low as 15.9%.⁸ Our current poor understanding of NSCLC pathogenesis, insufficient early diagnostic biomarkers, and a lack of therapeutic targets all contribute to these unfavorable outcomes.⁹

Fatty acid synthase (FASN) is a multi-functional enzyme that catalyzes the biosynthesis of palmitate in a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent manner.¹⁰ Most normal cells primarily import lipids from the extracellular milieu and do not have a strict requirement for FASN activity.^11,12 Nevertheless, FASN is ubiquitously expressed by normal cells in adult tissues, albeit at low to moderate levels. In contrast, tumor cells have an increased requirement of lipids for membrane biosynthesis, protein modification, and signaling and are more dependent on de novo palmitate synthesis catalyzed by FASN.^13,14 Accordingly, FASN is overexpressed in many solid and hematopoietic tumors, including those of breast, ovarian, prostate, colon, lung, and pancreatic origins.^15–19 Furthermore, FASN expression in tumors increases in a stage-dependent manner and is associated with diminished patient survival.^{15,17,18,20,21} Meanwhile, the overexpression of genes involved in de novo lipogenesis is associated with prognosis for multiple cancer types including lung cancers. In addition, inhibition of FASN has anti-tumor effects in biologically diverse preclinical tumor models. These models provide mechanistic and pharmacological evidence that FASN inhibition represents a promising therapeutic strategy for multiple cancers, including those expressing mutant K-Ras, ErbB2, c-Met, and PTEN.²² All these reports suggest that FASN could serve as a diagnostic biomarker and therapeutic target for the treatment of NSCLC.

Long non-coding RNAs (lncRNAs) are widely defined as transcripts longer than 200 nucleotides without open reading frames.^23,24 Dong et al.²⁵ found lncRNA, GAS5, enhanced gefitinib-induced cell death in innate epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor–resistant LAD cells. Lung cancer cell elevated lncRNA HOX transcript antisense RNA (HOTAIR) represses gene expression through recruitment of chromatin modifiers, and the high expression of HOTAIR correlates with metastasis and poor prognosis. Moreover, HOTAIR promotes proliferation, survival, invasion, metastasis, and drug resistance in lung cancer cells.²⁶ The development of technological methods such as lncRNA microarrays and RNA sequencing has enabled identification of dysregulated lncRNAs that function as oncogenes or tumor suppressor genes in NSCLC.⁹ Some of these dysregulated lncRNAs are associated with different stages of NSCLCs, some are specifically overexpressed in a particular lung cancer subtypes, and others play roles in drug resistance. These findings suggest important roles for lncRNAs in the pathogenesis of NSCLC.⁹ However, only a small number of lncRNAs have been well characterized, and the functions of most lncRNAs remain unknown.

HAGLR, or HOXD-AS1, contains eight exons and its transcript is a novel lncRNA. It is transcribed from the HOXD cluster on human chromosome 2q31.2 in an antisense manner.²⁷ In addition, HAGLR is closely interrelated with the expression of HOXD3.²⁸ A high level of HAGLR expression is associated with neuroblastoma progression and dysregulation of phosphoinositide 3-kinase (PI3K)/Akt signaling, with this pathway promoting oncogenesis via inhibition of apoptosis.²⁹ Moreover, multiple genes are influenced by HAGLR and these genes may serve as biomarkers of neuroblastoma recurrence and affect patient survival.³⁰ HAGLR is upregulated in neuroblastoma, adenocarcinoma, breast cancer, and bladder cancer. Moreover, it is closely associated with the progression and unfavorable prognosis of these cancers.^{27,28,30–32} HOXD is a member of the HOX cluster which regulates embryogenesis and organogenesis. HOX gene dysregulation occurs in multiple cancers including lung cancer.^33,34 However, whether HAGLR plays a role in NSCLC is unknown.

Our study aimed to investigate HAGLR expression in NSCLC and further explore its clinical significance and the molecular mechanisms involved. We identified increased HAGLR expression in NSCLC tissues and this high expression correlates clinicopathological features, such as tumor stage and survival rate. HAGLR expression levels positively associated with NSCLC cell proliferation. Furthermore, increased HAGLR expression may promote NSCLC cell cycling by enhancing de novo lipogenesis. Our results suggest that HAGLR may serve as an oncogene in the progression of NSCLC and represents a potential target for NSCLC treatment.

Materials and methods

Patients, tissue specimens and cell lines

A total of 60 pairs of primary NSCLCs and corresponding non-cancerous adjacent tissues were collected by Zhongshan Hospital, Fudan University, with appropriate informed consent obtained after institutional review board approval. All specimens were obtained from NSCLC patients who underwent resection without radiotherapy or chemotherapy before surgery and clinical data were collected from all patients at the same time.

Cell culture

NSCLC cell lines—A549, SPC-A1, NCI-H1975, NCI-H1703, and SK-MES-1—and BEAS-2B normal human bronchial epithelial cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA) in a 5% CO₂ humidified incubator at 37°C.

Construction of a HAGLR small interfering RNA lentiviral expression vector

Target sites for RNA interference of human HAGLR were identified in a previous work.²¹. The sequences used to construct human HAGLR-targeting and negative control small interfering RNA (siRNA) were 5′-GAAAGAAGGACCAAAGTAA-3′ (siLncR-1), 5′-GCACAAAGGAACAAGGAAA-3′ (siLncR-2), and 5′-GGGATCATGGGTGTCATCTAC-3′ (siNC). DNA oligonucleotides incorporating the short hairpin RNA (shRNA)-encoding sequences were then synthesized by Shanghai GenePharma Co., Ltd (Shanghai, China) and annealed into double strands by incubation in Annealing Buffer for RNA Oligos (Beyotime Institute of Biotechnology, Haimen, China). Following digestion with BamHI and EcoRI restriction endonucleases (TransGen Biotech Inc., Beijing, China), the double-stranded DNA molecules were inserted into the pGCSi-neo-GFP lentiviral vector. Successful insertion was confirmed by DNA sequencing. pGCSi-neo-GFP-HAGLR-shRNA/NC-shRNA plasmid DNA and packaging vectors were transiently transfected into human embryonic kidney 293T (HEK293T) cells (ATCC) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA; Thermo Fisher Scientific) according to the manufacturer’s instructions. At 48 h after transfection, supernatants containing lentiviral particles were collected and purified by ultracentrifugation at 70,000g at 4°C for 2 h. Viral titer was determined using the Lentivirus-Associated HIV p24 ELISA Kit (Cell Biolabs Inc., San Diego, CA, USA).

Cell transfection

For transfection, cultured cells were seeded into a six-well plate at a density of 1.0 × 10⁵ cells/well for 24 h. Lentiviral particles encoding shRNA targeting HAGLR or NC-shRNA were added into the medium and incubated for 24 h. After replacing the culture medium in each well, cells were incubated for a further 48 h prior to further analyses.

RNA extraction and real-time polymerase chain reaction

Total RNA was obtained from tissues or cells using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Complementary DNA (cDNA) was transcribed from 1 µg total RNA using the Reverse Transcription Kit (TaKaRa, Dalian, China). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed on the StepOne Plus system (Applied Biosystems, Foster City, CA, USA). Each reaction contained 2-µL cDNA, 0.5-µL forward primer, 0.5-µL reverse primer, 5-µL qPCR Mix (TaKaRa), and 3-µL deionized water. The 2^−ΔΔCt method was used to calculate relative expression levels. Expression level of β-actin was measured as the endogenous control. The primer sequences used were as follows: HAGLR forward 5′-GGCTCTTCCCTAATGTGTGG-3′, reverse: 5′-CAGGTCCAGCATGAAACAGA-3′; β-actin forward 5′-CGCTCTCTGCTCCTCCTGTTC-3′, reverse: 5′-ATCCGTTGACTCCGACCTTCAC-3′.

Free fatty acid quantification

Levels of free fatty acid were determined using the Free Fatty Acid Quantificaton Kit (MAK044; Sigma-Aldrich, St. Louis, MO, USA). Briefly, cells or tumors were divided equally, with one half lysed in 1% Triton X-100 in chloroform (w/v). The samples were centrifuged at 12,000g for 10 min to remove insoluble debris. The organic phase was collected and allowed to air-dry on a 50°C dry bath for 20 min. Samples were then vacuum dried for 30 min to remove traces of chloroform. Dried lipids were resuspended via vortexing in fatty acid assay buffer and were further quantified following the manufacturer’s instruction. The total protein weight was determined by Bradford assay. Values are reported as µM/20 mg protein.

MTT assay

For cell proliferation assays, SPC-A1 and NCI-1703 cells stably transfected with HAGLR shRNA or NC shRNA were detached with 0.25% trypsin (Boster Inc., Wuhan, China), seeded into 96-well plates at a density of 1000 cells/well in a final volume of 100 µL, and maintained in DMEM supplemented with 10% FBS. At different time points (24 h, 48 h, 72 h, 4 days, 5 days, 6 days, or 7 days after plating), 10-µL MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) solution (Sigma-Aldrich) was added to each well, and cells were incubated for an additional 4 h at 37°C. Blue formazan crystals were then dissolved in 100-µL dimethyl sulfoxide (Origen Biomedical, Austin, TX, USA), and the absorbance was measured at 490 nm using a microplate reader (ELx800; BioTek Instruments Inc., Winooski, VT, USA)

Flow cytometric analysis

Cells were harvested by trypsinization 72 h after transfection and then fixed in 70% ethanol overnight. Fixed cells were rehydrated in phosphate-buffered saline and subjected to propidium iodide (PI)/RNase staining followed by fluorescence-activated cell sorting analysis by BD FACSCalibur (Becton Dickinson, Mountain View, CA, USA). Data were analyzed using FlowJo analysis software (Tree Star, Ashland, OR, USA).

Western blot assay

Total protein from NSCLC tissues or cultured cells was extracted and quantified using the Bradford method. Approximately 20 µg of total protein from each sample was separated on a 12% gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). Primary antibodies for FASN (1:1000; Boster Inc.) and β-actin (1:2000; Boster Inc.) were separately incubated with membranes at 4°C overnight. After incubation with peroxidase-coupled secondary antibody for 2 h, protein bands were detected on a G: Box iChem Imager (Syngene, Cambridge, UK) using enhanced chemiluminescence reagent (TianNeng, Shanghai, China). Expression levels of ABCC3 were quantified by measuring β-actin levels as a loading control.

Transwell assay

A Transwell assay was performed to assess the invasiveness of SPC-A1 and NCI-H1975 cells using chambers with an 8.0-µm transparent polyethylene terephthalate membrane in 24-well plates (Corning Inc., Corning, NY, USA). Cells (2.0 × 10⁵ per chamber) were seeded into the upper chambers in 200 µL serum-free DMEM, and 500 µL of DMEM with 10% FBS was added to each lower chamber. Each condition was evaluated in triplicate. After 12 h, the cells on the top surface of the membrane were gently removed with cotton swabs. Cells that had passed through the filters were fixed in methanol for 15 min and stained with hematoxylin (OriGene China, Beijing, China) for 20 min, air-dried, and photographed (Olympus Stream Image Analysis Software; Olympus Corporation, Tokyo, Japan). The invasive cells were counted using a microscope (magnification, ×200; CX31; Olympus Corporation) in five randomly selected visual fields.

Xenograft model

To examine the ability of tumor formation in vivo, cell lines stably transfected with HAGLR shRNA or NC-shRNA were injected into nude mice. Ten 4-week-old BALB/c male nude mice were purchased from the Vital River Laboratories (Beijing, China) and randomly assigned to one of two groups. All nude mice were bred and maintained at Vital River Laboratories under specific pathogen-free conditions. The research program, objectives, and animal use protocols were reviewed and approved by the Animal Ethical and Welfare Committee of Tsinghua University. Cell suspensions (100 µL) containing 1.0 × 10⁷ SPC-A1-HAGLR-shRNA or SPC-A1-NC-shRNA cells were injected subcutaneously into the right flank region of each nude mouse. Tumor size and mouse weight were measured every 4 days from day 8 following injection. On the day 28 following injection, all mice in the two groups were sacrificed by cervical dislocation and the primary tumors were removed from each mouse. Tumor size and tumor weight were measured and recorded. The tumor volume was calculated using the formula: volume = width² × length × 0.5.

Statistical analysis

SPSS version 18.0 for Windows was used for all analyses. Results are expressed as the mean ± standard error. The significance of differences between experimental groups was evaluated using χ² analysis. Survival analyses were performed using the log-rank test and Kaplan–Meier plots approach. Student’s t-test was used to compare other data. For all analyses, the level of significance was set at p < 0.05.

Results

HAGLR expression levels are upregulated in NSCLC tissues and cells positively correlate with significantly shorter survival of NSCLC patients

We first measured the relative HAGLR RNA expression in NSCLC data obtained from the GEO database to evaluate whether HAGLR is involved in NSCLC. HAGLR RNA levels were upregulated in NSCLC specimens compared with normal tissues (Figure 1(a); p < 0.01), suggesting that high levels of HAGLR in NSCLC tissues may serve as an oncogenic function in NSCLC. Moreover, HAGLR expression levels were measured by RT-qPCR in five NSCLC cell lines—A549, SPC-A1, NCI-H1975, NCI-1703, and SK-MES-1—and BEAS-2B normal human bronchial epithelial cells. HAGLR expression was upregulated in all five NSCLC tumor cell lines, including 17.2- and 23.4-fold increases in SPC-A1 and NCI-1703 cell lines, respectively (Figure 1(b)). We next investigated the relationship between HAGLR expression levels and patient clinical pathologic features. The patients were classified into low-HAGLR and high-HAGLR groups according to mRNA expression levels (Table 1). HAGLR expression levels significantly correlated with lymph node metastasis status (p = 0.001) and tumor stage (p = 0.003). Kaplan–Meier survival analysis was employed to evaluate OS of NSCLC patients, and the log-rank test revealed that the HAGLR-high groups had a significantly lower OS than the HAGLR-low group (Figure 1(c); p = 0.0239)

Figure 1.

High expression of HAGLR correlates with poor survival of NSCLC patients. (a) Relative levels of HAGLR RNA in 60 pairs of NSCLC tumors and corresponding normal lung tissues as determined by quantitative-RT-PCR. (b) HAGLR RNA levels in bronchial epithelial (BEAS-2B) and NSCLC (A549, SPC-A1, NCI-H1975, NCI-H1703, and SK-MES-1) cell lines. (c) Kaplan–Meier analysis of patient survival based on HAGLR expression level.

Table 1.

Relationship between HAGLR expression levels and clinicopathological features in NSCLC specimens.

	HAGLR expression
	High	Low	p
Gender			0.793
Male	12	19
Female	10	19
Age (years)			0.789
<60	9	18
⩾60	13	20
Size of tumor (cm)			0.589
⩽3	11	15
>3	11	23
Histology			0.234
Squamous cell carcinoma	8	8
Adenocarcinoma	14	30
Lymph node metastasis			0.001*
N0	5	26
N1–3	17	12
pTNM stages			0.003*
I–II	10	32
III–IV	12	6

NSCLC: non–small cell lung cancer; pTNM: pathological tumor-node-metastasis.

Statistical significance (p < 0.05).

Knockdown of HAGLR inhibits FASN in NSCLC cells

While HAGLR expression levels are closely associated with the progression and prognosis of multiple tumor types, the mechanisms involved are incompletely understood. Therefore, we next investigated whether there was any association between HAGLR levels and FASN expression in 60 pairs of clinical NSCLC specimens. HAGLR levels were positively correlated with FASN (p = 0.0344; Figure 2(a)). We then selected the SPC-A1 and NCI-1703 cells lines to further investigate the association between HAGLR and FASN, as these cell lines had the highest levels of HAGLR upregulation. SPC-A1 and NCI-1703 cells were transfected with siLncR-1, siLncR-2, or siNC to investigate whether knockdown of HAGLR could inhibit FASN expression in NSCLC tumor cells. The relative expression level of HAGLR was measured by RT-qPCR at 48 h after transfection. Figure 2(b) shows that the relative level of HAGLR in NSCLC cells was significantly decreased by both siLncR-1 and siLncR-2; siLncR-1 reduced HAGLR levels by 76.1% in SPC-A1 cells and 69.1% in NCI-H1703 cells, while siLncR-2 reduced levels by 80.3% in SPC-A1 cells and 77.4% in NCI-H1703 cells. The relative expression levels of HAGLR following transfection with siLncR-1 was reduced by 67.2% in SPC-A1 cells and 64.3% in NCI-1703 cells, while siLncR-2 reduced HAGLR by 69.2% in SPC-A1 cells and 68.5% in NCI-1703 cells. Consistently, western blotting identified decreased protein levels of FASN in SPC-A1 and NCI-1703 cells following knockdown of HAGLR (Figure 2(c)). Furthermore, knockdown of HAGLR significantly decreased the cellular free fatty acid content of both SPC-A1 and NCI-1703 cells (Figure 2(d)). Taken together, these results show that knockdown of HAGLR inhibits FASN in NSCLC tumor cells.

Figure 2.

Downregulation of HAGLR leads to decreased de novo lipogenesis in NSCLC cells. (a) Analysis of the correlation between HAGLR and FASN expression levels in 60 NSCLC tumor tissues. (b) RNA interference–mediated inhibition of HAGLR and (c) evaluation of FASN protein and (d) cellular free fatty acid levels in SPC-A1 and NCI-H1703 cells.

Downregulation of HAGLR inhibits NSCLC cell proliferation and invasiveness

FASN is a vital enzyme in tumor cell biology, and its overexpression by tumor cells is associated with poorer patient prognosis and resistance to cancer therapy. However, FASN inhibition prevents tumor cell growth, blocks proliferation, and impairs survival. Therefore, we next investigated the effect of HAGLR on NSCLC tumor cell proliferation. SPC-A1 and NCI-1703 cells were transfected with siLncR-1, siLncR-2, or siNC, and an MTT assay was performed. No significant differences were observed within 48 h; however, the proliferative capacity of siLncR-1- and siLncR-2-transfected cells was markedly inhibited at 72 h when compared with NC cells (Figure 3(a)). Consistently, flow cytometry analysis revealed that the percentage of G1 phase cells was increased by approximately 11.2% (p < 0.05) and 13.5% (p < 0.05) following transfection with siLncR-1 and siLncR-2, respectively (Figure 3(b)). These data suggest that downregulation of HAGLR inhibited the proliferation of NSCLC cells through cell cycle arrest at G1.

Figure 3.

Decreased HAGLR levels inhibits proliferation and invasion in NSCLC cells. (a) MTT assay evaluating NSCLC proliferation following knockdown of HAGLR. (b) FACS and (c) transwell assays following knockdown of HAGLR in NSCLC cells to investigate cell cycle and invasion. (d) Western blots depicting p21 and MMP-9 protein levels changes in HAGLR-knockdown stable NSCLC cell lines.

We next evaluated the relationship between HAGLR and the invasive capacity of NSCLC cells using a transwell assay. The number of cells that crossed the filters was significantly decreased in both the siLncR-1 and siLncR-2 groups compared with the siNC group (p < 0.01; Figure 3(c)).

We further investigated the molecular mechanisms by which HAGLR regulates NSCLC cell cycle and invasiveness using western blotting. p21 was upregulated and matrix metalloproteinase protein (MMP)-9 was downregulated when HAGLR was knocked down (Figure 3(d)). These results show that the inhibition of NSCLC cell proliferation and invasion that follows HAGLR knockdown may result from dysregulation of p21 and MMP-9.

Knockdown of HAGLR in NSCLC cells inhibits xenograft tumor growth

We next investigated whether HAGLR expression could promote NSCLC progression and enhance NSCLC cell tumorigenicity using an in vivo tumor model. We used an shRNA lentiviral knockdown system to generate two stable cell lines, SPC-A1-siHAGLR (lncR-KD) and SPC-A1-siNC (NC), which were injected subcutaneously into the right flank of nude mice (n = 5 per group). Tumor volumes were measured every 3 days until day 33 following injection. Tumors from NC cells grew faster than those formed by lncR-KD cells (Figure 4(a)). In addition, the mean tumor weight of the lncR-KD group was significantly lower compared with that of the NC group (Figure 2(b); p < 0.01). Tumor-bearing mice were sacrificed on day 33 following injection, and tumor tissues were removed and photographed. Tumor size in the lncR-KD group was markedly smaller than that of the NC group (Figure 4(b)). We next used RT-qPCR to show that the relative level of HAGLR expression in lncR-KD-derived tumor tissues was significantly decreased compared with that in NC-derived tissues (Figure 4(c)). These results are consistent with those of our in vitro experiments and suggest that HAGLR regulates NSCLC tumor cell proliferation, invasion, and tumorigenic ability.

Figure 4.

HAGLR suppression inhibits NSCLC tumorigenicity and de novo lipogenesis in nude mice. (a) Thirty-three-day in vivo tumor growth curve of tumor xenografts derived from HAGLR-knockdown and control SPC-A1 cells. (b) Photographs and average tumor weight of the control and HAGLR-knockdown xenografts at the end point. (c) HAGLR expression in knockdown and control tissues as determined by RT-qPCR. (d) Western blotting and (e) free fatty acid assays of p21 and MMP-9 protein levels and de novo lipogenesis activity in xenograft tumor tissues.

Finally, we investigated whether the inhibition of xenograft tumor growth by knockdown of HAGLR in NSCLC tumor cells was associated with FASN inhibition. Western blotting showed that the protein levels of FASN and p21 were significantly decreased in tissues of lncR-KD-derived tumors when compared with NC-derived tumors (Figure 4(d)). Consistently, the cellular free fatty acid content of lncR-KD tumor tissues significantly decreased from 65 µM/20 mg protein to 23 µM/20 mg protein when compared with NC group (Figure 4(e); p < 0.01).

Discussion

HAGLR expression was upregulated in NSCLC specimens compared with normal tissues, with HAGLR levels in five NSCLC cell lines elevated 2- to 20-fold. Furthermore, Kaplan–Meier survival analysis revealed that high HAGLR expression was linked to poor OS in NSCLC patients (p = 0.0239). We also explored the biological function of HAGLR in NSCLC progression using a lentivirus-mediated shRNA expression system to disrupt HAGLR expression in NSCLC cells. Knockdown of HAGLR expression significantly suppressed the proliferation and invasion of both SPC-A1 and NCI-H1703 cells in vitro and effectively reduced SPC-A1 cell oncogenicity in vivo. Taken together, these findings indicate that HAGLR participates in the development and progression of NSCLC. Therefore, an elevated HAGLR expression level may be a predictor of poor prognosis for NSCLC patients.

Previous reports suggest that inhibition of de novo palmitate synthesis via disruption of FASN is an approach to cancer therapy that is as-yet unproven but has a strong biological rationale.^22,35,36 FASN expression increases with tumor progression and is associated with chemoresistance, tumor metastasis, and diminished patient survival in numerous tumor types including lung cancer.²² LncRNAs are functionally associated with essential biological processes, including RNA splicing, protein localization, and fatty acid metabolic process.³⁷ Here, we have demonstrated that repression of FASN follows silencing of HAGLR. Emerging evidence indicates that FASN play key roles during tumor genesis and development and may serve as a therapeutic target in multiple human cancers.^38–40 Our present findings indicate that HAGLR levels may regulate FASN activity, though further research is necessary to clarify the precise relationship between HAGLR and FASN.

Our study reveals that HAGLR expression is upregulated in the majority of NSCLC tumors and is closely associated with tumor lymph node metastasis status, stage, and poor OS in NSCLC patients. Furthermore, HAGLR overexpression promotes NSCLC progression at least in part by activating FASN, MMP-9, and p21. Therefore, high expression of HAGLR may a drugable target for clinical use and useful biomarker for diagnosis and prognosis of NSCLC.⁴¹

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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Increased HAGLR expression promotes non–small cell lung cancer proliferation and invasion via enhanced de novo lipogenesis

Abstract

Keywords

Introduction

Materials and methods

Patients, tissue specimens and cell lines

Cell culture

Construction of a HAGLR small interfering RNA lentiviral expression vector

Cell transfection

RNA extraction and real-time polymerase chain reaction

Free fatty acid quantification

MTT assay

Flow cytometric analysis

Western blot assay

Transwell assay

Xenograft model

Statistical analysis

Results

HAGLR expression levels are upregulated in NSCLC tissues and cells positively correlate with significantly shorter survival of NSCLC patients

Knockdown of HAGLR inhibits FASN in NSCLC cells

Downregulation of HAGLR inhibits NSCLC cell proliferation and invasiveness

Knockdown of HAGLR in NSCLC cells inhibits xenograft tumor growth

Discussion

Footnotes

Declaration of conflicting interests

Funding

References