JAK2 variations and functions in lung adenocarcinoma

Abstract

Lung cancer ranks as the first most common cancer and the first leading cause of cancer-related death in China and worldwide. Due to the difficulty in early diagnosis and the onset of cancer metastasis, the 5-year survival rate of lung cancer remains low. JAK2 has emerged as pivotal participant in biological processes, often dysregulated in a range of cancers. Recently our study found that JAK2 might play an important role in lung cancer pathogenesis. While our understanding of JAK2 in the onset and progression of lung cancer is still in its infancy, there is no doubt that understanding the variations and functions of JAK2 will certainly secure strong biomarkers and improve treatment options for lung cancer patients. The expression level of JAK2 mRNA was assayed using RT-PCR. JAK2 mutations and amplification were detected using next-generation sequencing (NGS). The shRNA and overexpression plasmids of JAK2 were conducted. MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazoliumbromide) assay, Trans-well migration and Matrigel invasion assay were conducted to study the proliferation, migration and invasion abilities of lung adenocarcinoma cells independently. We found that JAK2 mRNA was up-regulated in lung adenocarcinoma tissues when compared with their adjacent non-tumor tissues, and was associated with lymph node metastasis (p < 0.05). JAK2 V617F and N30S mutations and JAK2 amplification were detected by NGS in plasmid samples in patients with lung adenocarcinoma. Downregulation of JAK2 inhibited the proliferation, migration and invasion abilities of lung adenocarcinoma A549 cells. Moreover, overexpression of JAK2 induced the proliferation, migration and invasion abilities of A549 cells. Thus, the up-regulation, mutation and amplification of JAK2 detected in lung adenocarcinoma may participate in lung cancer progression by regulating cancer cells’ proliferation, migration and invasion.

Introduction

Lung cancer ranks as the first most common cancer and the first leading cause of cancer-related death in China and worldwide. ¹ The non-small-cell lung cancer (NSCLC) is the most prominent subgroup accounting for approximately 80% of all lung cancer cases, which includes adenocarcinomas, squamous cell carcinomas, and large-cell carcinomas. ² Despite the recent advances in the strategies of lung adenocarcinoma diagnosis and treatment recently, such as epidermal growth factor receptor (EGFR) and anaplastic lymphoma receptor tyrosine kinase (ALK) gene detection and correlated targeted therapy, the prognosis remains poor, with a 5-year overall survival rate of about 11%. ³ This is mainly due to the incidence of cancer metastasis and the development of drug resistance. The underlying molecular mechanisms of lung adenocarcinoma progression and drug resistance have not been thoroughly elucidated. Therefore, identifying new diagnostic, prognostic, and resistance biomarkers along with therapeutic targets is still of paramount importance for lung adenocarcinoma research.

Constitutive activation of STAT3 is common in many solid tumors including lung adenocarcinoma and is required for efficient cellular transformation by many well-characterized oncogenes.^4,5 It has been demonstrated that activation of STAT3 is commonly achieved by somatic mutations of JAK2 in hematologic malignancies, but similar mutations are not often found in solid tumors including lung cancer.^6–10 It has been over a decade since JAK2 was first cloned. ¹¹ JAK2 is expressed in nearly every tissue and associated with many pathological implications. Although it is evident that JAK2 acts as an oncogene in both myeloproliferative disorders and some solid tumors,^12–16 no mutations were detected and no direct involvement of JAK2 was found in lung cancer migration, invasion, or metastasis.

In this study, JAK2 variations including dysregulation, mutation, and amplification were found in lung adenocarcinoma tissues and plasmid samples from NSCLC patients. Moreover, dysregulation of JAK2 could influence the proliferation, migration, and invasion abilities of lung adenocarcinoma A549 cells. Given the above results, JAK2 might have therapeutic potential in lung adenocarcinoma treatment.

Materials and methods

Clinical samples

Clinical lung adenocarcinoma specimens and their pair-matched non-malignant lung tissue samples from 40 patients undergoing lung cancer resection were provided by Zhejiang Cancer Hospital (Hangzhou, China). They included 15 males and 25 females with an age range of 40–75 years with a median of 55 years. All the samples were collected with written informed consent from the patients. Both lung tumor tissues and adjacent non-tumor lung tissues collected after surgery were divided into two parts. One was frozen in liquid nitrogen immediately for further use; the other part was stored in formalin for pathology analysis.

Cell culture

The lung adenocarcinoma cell line A549 was purchased from Zhejiang University School of Medicine (Hangzhou, China). This cell line was maintained at 37°C in an atmosphere of 5% CO₂ in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin (Gibco BRL) in 25-mL culture flasks.

Cell proliferation assay

To determine the effect of JAK2 on proliferation of lung cancer cell line A549, 4 × 10³ cells were transfected with pS-JAK2 or flag-JAK2 or control plasmid (pS-con or flag-con) using lipo2000 transfection agent or with mock control following manufacturer’s protocol in 96-well plate. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) assay was performed at 0, 24, 48, 72, and 96 h post transfection. ¹⁷ The absorbance of the samples was measured with a spectrophotometer reader at 490 nm. Each assay was performed in triplicate and repeated three times independently.

Migration and invasion assay

Cells were transfected with pS-JAK2 or flag-JAK2 or control plasmid (pS-con or flag-con) using Lipofectamine 2000 transfection agent following manufacture’s protocol in 24-well plates. Transwell migration assay and Matrigel invasion assay were performed separately using 24-well Transwell inserts with 8 µm pore size (Corning Costar Corp.) 24 h after transfection. For Transwell migration assay, 2 × 10⁴ A549 cells suspended in 100 µL corresponding culture medium without FBS were loaded into the top chamber of Transwell insert with non-coated membrane. ¹⁵ For Matrigel invasion assay, 4 × 10⁴ A549 cells were plated in 100 µL serum-free medium in the upper Matrigel-coated chamber instead. ¹⁵ In both assays, the bottom chamber was containing 600 µL medium with 20% FBS. Cells were then allowed to migrate or invade for 12 h at 37°C. The cells that migrated or invaded into the bottom chamber were fixed, stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:1000), visualized under phase-contrast microscope, and photographed. Total number of migrated or invaded cells was counted by Image-Pro Plus 6.0 (IPP) software. Count and characterize objects using over fifty manual and automatic measurement tools including areas, perimeters, lengths, roundness, major and minor axes, angles, centroids, holes, and population density. Tag objects of interest and easily sort by size or other measurement parameters. All experiments were independently repeated at least three times.

Construction of plasmids

For ectopic expression of FLAG-tagged JAK2, human JAK2 with coding region was cloned into pCMV-Tag 2C vector. ¹⁶ To deplete endogenous JAK2 by RNA interference (RNAi), the oligo-containing complementary hairpin sequences were synthesized and cloned into pSilencer 4.1-CMV neo vector (Ambion). ¹⁶ The targeting sequence of JAK2 gene is 5′-AAC TCT ATC AGC TAC AAG ACA-3′, corresponding to the region 604–624 relative to the first nucleotide of the start codon. A circular pSilencer 4.1-CMV neo vector that expresses a hairpin small interfering RNA (siRNA) with limited homology to any known sequence was used as a negative control. All constructs were confirmed by sequencer used.

RNA extraction

Total RNA from lung cancer or normal tissue samples was extracted using the TRIzol Reagent Kit (Invitrogen) following the manufacture’s protocol. RNA concentrations and quality were determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies) and gel analysis.

Quantitative real-time polymerase chain reaction analysis

The expression of JAK2 messenger RNA (mRNA) was assayed with specific primers on tissue samples. Reverse transcription reaction was carried out starting from 10 ng of total RNA using the looped primers. Real-time polymerase chain reaction (RT-PCR) was performed using the standard protocol on ABI7500 real-time PCR detection system. ¹⁶ The ΔΔCt method for relative quantization was used to determine mRNA expression. The Ct is the fractional cycle number at which the fluorescence of each sample passes the fixed threshold. The ΔCt was calculated by subtracting the Ct of small nuclear RNA (snRNA) U6 (RNU6B; Applied Biosystems) from the Ct of the mRNA. The ΔΔCt was calculated by subtracting the ΔCt of the reference sample (paired non-tumor tissue for surgical samples) from the ΔCt of each sample. Fold change was determined as 2^−ΔΔCt.

Next-generation sequencing

Next-generation sequencing (NGS) was performed by Geneseep. Tumor genomic DNA was extracted from formalin-fixed and paraffin-embedded (FFPE) tumor tissues, and the fragment DNA was generated with Bioruptor from patients’ plasma samples (Bioruptor UCD-200; Diagenode) following manufacturer’s protocol. Libraries were constructed using the KAPA Hyper DNA Library Prep Kit (KK8504; KAPA Biosystems). Finally, dual-indexed sequencing libraries were PCR amplified with KAPA HiFi HotStart ReadyMix (KK2602; KAPA Biosystems) for 4–6 cycles and then cleaned up by purification beads (AxyPrep Fragment Select-I kit, 14223162; Corning Costar Corp.). Library concentration and quality were determined by the Qubit 3.0 system (Invitrogen) and Bioanalyzer 2100 (Agilent HS DNA Reagent, 5067-4627; Agilent).

The 5′-biotinylated probe solution was used as the capture probes. The baits targeted 14 lung cancer–related genes. A volume of 1 µg of each fragment sequencing library was mixed with 5 µg of salmon sperm DNA, 5 µg of human Cot-1 DNA, and 1 unit adaptor-specific blocker DNA in hybridization buffer, heated for 10 min at 95°C, and held for 5 min at 65°C in the thermocycler. The capture probes were added to the mixture in 5 min, and the solution hybridization was performed for 16–18 h at 65°C. After hybridization was complete, the captured targets were selected by pulling down the biotinylated probe/target hybrids using streptavidin-coated magnetic beads, and off-target library was removed using wash buffer. The PCR master mix was directly added to amplify (6–8 cycles) the captured library from the washed beads. After that, the samples were purified by AMPure XP beads, quantified by quantitative PCR (qPCR; KAPA), and sized on Bioanalyzer 2100 (5067-4627; Agilent HS DNA Reagent; Agilent). Libraries were normalized to 2.5 nM and pooled. Finally, the library was sequenced as paired 75-bp reads on Illumina HiSeq 4000 according to manufacturer’s instructions.

Statistical analyses

Data are represented as mean ± standard error (SE) of three independent experiments. Student’s t-test and χ² test were performed to determine statistical significance; p < 0.05 was considered statistically significant.

Results

Expression level of JAK2 mRNA in lung adenocarcinoma samples

The relative expression level of JAK2 mRNA in lung adenocarcinoma samples showed a range of 2.2–4.8 fold changes as compared with their adjuvant non-tumor tissues with a median of 3.1-fold change. Patients were divided into two groups (relatively high expression level group and relatively low expression level group) according to the expression level of JAK2 mRNA. No significant association was encountered between JAK2 expression level and clinicopathological features except for an association with lymph node (LN) metastasis (p = 0.025; Table 1).

Table 1.

Relationship between the expression level of JAK2 mRNA and clinical and pathological characteristics of lung adenocarcinoma patients.

Parameter	JAK2 mRNA expression		p
	Relatively high expression level (n = 20)	Relatively low expression level (n = 20)
Gender			0.744
Male	8	7
Female	12	13
Age			0.507
⩽65	6	8
>65	14	12
Differentiation			0.785
High	5	6
Middle	7	5
Low	8	9
Lymph node metastasis			0.025
Absent	5	12
Present	15	8
AJCC/UICC stage			0.803
I	6	7
II	8	6
III	6	7
IV	0	0

AJCC: American Joint Committee on Cancer; UICC: Union for International Cancer Control; mRNA: messenger RNA.

JAK2 mutations and amplification in lung adenocarcinoma

In the fourth quarter of 2016, the samples of 1100 patients (including FFPE tumor tissues and pair-matched plasma samples) with lung adenocarcinoma were detected by NGS searching for driver gene mutations. JAK2 variations were detected in 10/1100 (0.9%) lung adenocarcinoma patients’ plasma samples, but not FFPE tumor tissue samples, including mutations and amplification. Among the 10 patients, 6 were detected with JAK2 V617F mutation, 1 with JAK2 N30S mutation, and 3 with JAK2 amplification (Figure 1).

Figure 1.

The IGV printscreen of JAK2 mutations. (a) The IGV printscreen of JAK2 V617F mutation. (b) The IGV printscreen of JAK2 N30S mutation.

Impact of JAK2 expression on adenocarcinoma cell proliferation

To explore the role of JAK2 in lung carcinogenesis, we first examined the effect of JAK2 RNAi on the proliferation of A549 cell. The cells were transfected with either pS-JAK2 or control plasmid (pS-con). The western blot analysis confirmed that the expression of JAK2 was significantly downregulated in cells transfected with pS-JAK2 compared with control plasmid (data not shown). MTT assays showed that downregulation of JAK2 significantly inhibited the proliferation of A549 cells (Figure 2(a)).

Figure 2.

Impact of JAK2 expression on adenocarcinoma cell proliferation. (a) MTT assays showed that downregulation of JAK2 by pS-JAK2 significantly inhibited the proliferation of A549 cells. (b) MTT assays showed that overexpression of JAK2 by flag-JAK2 significantly induced the proliferation of A549 cells.

Then, we examined the effect of JAK2 overexpression on the proliferation of A549 cell. The cells were transfected with either flag-JAK2 or control plasmid (flag-con). The western blot analysis confirmed that the expression of JAK2 was significantly increased in cells transfected with flag-JAK2 compared with control plasmid (data not shown). MTT assays showed that overexpression of JAK2 significantly induced the proliferation of A549 cells (Figure 2(b)).

Impact of JAK2 downregulation on lung cancer cell migration and invasion

To explore the potential role of JAK2 in lung cancer metastasis, we first examined the effect of JAK2 RNAi on the migration of A549 cells. Cells were transfected with either pS-JAK2 or control plasmid (pS-con). The Transwell migration assay showed that downregulation of JAK2 greatly inhibited the migration of A549 cells (Figure 3). We further employed Matrigel invasion assay and found that downregulation of JAK2 also inhibited the invasive properties of A549 (Figure 4).

Figure 3.

Downregulation of JAK2 suppressed the migration of A549 cells. A549 cells transfected with pS-JAK2 or control plasmid (pS-con) were subjected to Transwell migration assay. (a) Representative fields of invasive cells on the underside of membrane which were fixed and stained with DAPI. (b) Total number of migrated cells on the underside of membrane was counted by IPP 6.0 software (*p < 0.05).

Figure 4.

Downregulation of JAK2 suppressed the invasion of A549 cells. A549 cells transfected with pS-JAK2 or control plasmid (pS-con) were subjected to Matrigel invasion assay. (a) Representative fields of invasive cells on the underside of membrane which were fixed and stained with DAPI. (b) Total number of migrated cells on the underside of membrane was counted by IPP 6.0 software (*p < 0.05).

Impact of JAK2 overexpression on lung cancer cell migration and invasion

We then examined the effect of JAK2 overexpression on the migration and invasion of A549 cells. Cells were transfected with either flag-JAK2 or control plasmid (flag-con). The Transwell migration assay showed that overexpression of JAK2 greatly induced the migration of A549 (Figure 5). We further employed Matrigel invasion assay and found that overexpression of JAK2 also promote the invasive properties of A549 (Figure 6). Collectively, these results indicate that overexpression of JAK2 is sufficient to induce both the migration and invasion abilities of lung adenocarcinoma cells.

Figure 5.

Overexpression of JAK2 induced the migration of A549 cells. A549 cells transfected with flag-JAK2 or control plasmid (flag-con) were subjected to Transwell migration assay. (a) Representative fields of invasive cells on the underside of membrane which were fixed and stained with DAPI. (b) Total number of migrated cells on the underside of membrane was counted by IPP 6.0 software (*p < 0.05).

Figure 6.

Overexpression of JAK2 induced the invasion of A549 cells. A549 cells transfected with flag-JAK2 or control plasmid (flag-con) were subjected to Matrigel invasion assay. (a) Representative fields of invasive cells on the underside of membrane which were fixed and stained with DAPI. (b) Total number of migrated cells on the underside of membrane was counted by IPP 6.0 software (*p < 0.05).

Discussion

In the last decade, there was significant progress in understanding the underlying molecular pathology and marked heterogeneity of lung cancer, especially adenocarcinoma. Multiple signaling pathways, including EGFR and ALK, have been identified as driver genes that associated with malignant transformations. In fact, the vast majority of lung cancer patients have no known driver mutations detected, and they are still treated with standard cytotoxic chemotherapy. Moreover, acquired resistance is common in patients with a known driver mutation. Thus, we are still challenged in the goal to elucidate the underlying molecular mechanism of lung adenocarcinoma development and progression.

The JAK/STAT pathway is required for cell survival and differentiation in many cancers. ¹⁸ JAK2 is a member of the Janus family of cytoplasmic non-receptor tyrosine kinases. ¹² Constitutive activation of STAT3 seems to be a common characteristic in many solid tumors including NSCLC. And STAT3 activation is frequently stimulated by JAK2 somatic mutations in hematologic malignancies, which are not common in solid tumors. V617F mutation of JAK2 is detected only in patients with polycythemia vera, essential thrombocythemia, and myelofibrosis, resulting in activation of tyrosine kinase, phosphorylation of STAT3, and expression of target genes.^{6–9,19–21} Moreover, JAK2 inhibitor TG101348 has been shown to inhibit cell proliferation of primary hematopoietic cells and erythroblastic leukemia (HEL) cell line that harbors the JAK2 V617F mutation.^22,23 However, little is known about the role of JAK2 in solid tumors, and reports failed to detect JAK2 mutation in solid tumors including lung cancer. ¹⁰ There is only a rare case depicting the association of JAK2 V617F mutation with myeloproliferative, lymphoproliferative, and solid neoplasms. ²⁴

Here, for the first time, we provide evidence that upregulation of JAK2 is associated with lung adenocarcinoma LN metastasis. Furthermore, downregulation of JAK2 was found to significantly suppress the proliferation, migration, and invasion of lung adenocarcinoma cells. Contrarily, overexpression of JAK2 could significantly induce proliferation, migration, and invasion of lung adenocarcinoma cells. These data suggest that JAK2 may play a role in lung carcinogenesis.

More interestingly, JAK2 mutations and amplification were also detected in lung adenocarcinoma patients in our study. Consistent with previous studies, we did not detect the JAK2 mutation in lung cancer tissue samples but in the plasma samples from patients with lung adenocarcinoma using NGS method. Because of the small sample size and the low detection rate, we could not analyze the association between JAK2 mutation and the clinical characteristics of lung adenocarcinoma. But combining our results with the other study results, we hypothesize that JAK2 mutation might be associated with lung cancer progression, poor prognosis, and tyrosine kinase inhibitor (TKI) resistance.

JAK2 has been demonstrated to be involved in EGFR-TKI resistance in lung adenocarcinoma and JAK2 inhibitors could overcome EGFR-TKI resistance in NSCLC cells with mutated EGFR.^25–29 Studies indicate that treatment of NSCLC cells with the JAK2 inhibitors could suppress growth in soft agar and xenograft assays. ²⁸ Developing new agents to overcome the EGFR-TKI resistance would be important for long-term treatment in NSCLC patients, JAK2 might be one of the potential targets. Therefore, targeting JAK2/STAT3 may be a new treatment approach in NSCLC patients with EGFR-TKIs resistance.

Targeting JAK2 has emerged as an attractive strategy of novel drug development because it is mutated in many patients with myeloproliferative disorders. ³⁰ Most of the JAK2-directed drug discovery efforts are in early/mid-stage of clinical development or are yet to reach the clinic. Of these small-molecule inhibitors, lestaurtinib and INCB-18424 are currently being evaluated in phase II clinical trials. ³⁰ Thus, it will be very interesting to evaluate the effectiveness of these molecules in the treatment of lung cancer patients who harbor JAK2 mutations.

In addition, JAK2 amplification was detected in lung adenocarcinoma patient samples in our study. However, its mechanism and clinical significance are still unclear. Recently, it was found that JAK2 gene amplification might be correlated with genetic amplification of the PD-L1 gene. ³¹ In this study, they found that the lung cancer cell line HCC4006 harbored both JAK2 and PD-L1 amplification. Expression of the PD-L1 protein was significantly reduced by the JAK2 inhibitor TG101348. Their data suggest that expression of PD-L1 protein is upregulated by the simultaneous amplification of the PD-L1 and JAK2 genes through JAK-STAT signaling in NSCLC. Thus, it also will be interesting to further explore the potential involvement of JAK2 in lung cancer immune checkpoint therapy.

In summary, our results indicate that the dysregulation, mutation, and amplification of JAK2 are associated with lung adenocarcinoma progression, targeting JAK2 alone or in combination with other molecular targets, including EGFR-TKI or PD-L1, and could have potential value for lung cancer research, to overcome drug resistance, and for therapy in the future.