HIF‐1α is necessary for activation and tumour‐promotion effect of cancer‐associated fibroblasts in lung cancer

Abstract Cancer‐associated fibroblasts (CAFs) activation is crucial for the establishment of a tumour promoting microenvironment, but our understanding of CAFs activation is still limited. In this study, we found that hypoxia‐inducible factor‐1α (HIF‐1α) was highly expressed in CAFs of human lung cancer tissues and mouse spontaneous lung tumour. Accordingly, enhancing the expression of HIF‐1α in fibroblasts via hypoxia induced the conversion of normal fibroblasts into CAFs. HIF‐1α‐specific inhibitor or HIF‐1α knockout (KO) significantly attenuated CAFs activation, which was manifested by the decreased expression of COL1A2 and α‐SMA. In vivo, during tumour formation, the expression of Ki‐67 and proliferating cell nuclear antigen (PCNA) in the tumour tissue with HIF‐1α KO fibroblasts was significantly lower than that of normal fibroblasts. Moreover, HIF‐1α in fibroblasts could activate the NF‐κB signalling pathway and enhance a subsequent secretion of CCL5, thus promoting the tumour growth. In conclusion, our results suggest that HIF‐1α is essential for the activation and tumour‐promotion function of CAFs in lung cancer (LC). And targeting HIF‐1α expression on CAFs may be a promising strategy for LC therapy.


| INTRODUC TI ON
Lung cancer (LC) is characterized by high morbidity and mortality among cancers worldwide. The latest research estimates that by 2020, the 5-year survival rate of LC will be only 19%, second only to pancreatic cancer and liver cancer. 1 Because of the rapid proliferation and early metastasis of LC cells, about 57% of the LC patients were diagnosed at late clinical stage and the 5-year survival rate was low, only 5%. 2 Therefore, it is of great significance to explore the specific mechanism of LC progression. Recent research has shown that tumour microenvironment (TME) plays a crucial role in the development of LC. TME includes a variety of cell types, such as tumour cells, CAFs, vascular endothelial cells, lymphatic endothelial cells and immune cells. 3 As CAFs are the core elements of TME, the research of CAFs has been greatly expanded in recent years.
In tumour microenvironment, CAFs remodel the extracellular matrix composition and induce the infiltration of immune cells, which leads to the reprogramming of TME structure. These changes provide an appropriate environment for tumour growth. 4 In these processes, the versatility and plasticity of CAFs will generate a large number of subgroups, which can promote or inhibit tumour progression and invasion, depending on the circumstances. 5 Meanwhile, the various origins of CAFs also contribute to its biological heterogeneity. 3 So defining the distinctive roles and functions of each CAFs subpopulation will be helpful for understanding CAFs as a whole. CAFs are mainly derived from the activation of fibroblasts in the tumour stromal tissues. Fibroblasts can be activated into CAFs by some soluble signalling molecules in TME, thus leading to phenotype changes and functional genes being differentially expressed, thereby regulating the development of tumour. 3 However, it is unclear how to categorize CAFs into functional subtypes. It is known that fibroblasts can be activated by excessive biochemical activators from the TME such as transforming growth factorβ (TGFβ), sonic hedgehog (SHH), bone morphogenetic protein (BMP), platelet-derived growth factor (PDGF), IL-1 and IL-6. [5][6][7] Furthermore, tumour cell condition medium (CM) has been reported to activate the fibroblasts and increase ECM deposition. 8 After the fibroblasts activation, some protein markers are up-regulated, including α-smooth muscle actin (α-SMA/ACTA2), fibroblast-activating protein (FAP), platelet-derived growth factor receptorα (PDGFRα), fibroblast-specific protein 1 (FSP1/S100A4), collagen type Ⅰ Alpha 2 (COL1A2) and Podoplanin (PDPN), which are recognized as representative markers of CAFs. 9 These markers are lack of sensitivity and specificity, and there is no really highly specific CAFs marker. Therefore, it is necessary to find new molecular markers to identify the functional heterogeneity of CAFs, which is of great significance to the development of effective targeted therapy for LC. However, there are few studies on the activation and function of CAFs in LC.
The main function of lung is to absorb the O 2 for the whole body.
The oxygen content of lung is between 3.0 and 87.248 kPa. However, within NSCLC, the oxygen levels ranged from 0.0931 to 6.118 kPa.
Hypoxia is an important pathognomonic feature of many malignant tumours including LC. 10 With the rapid growth of LC cells, the oxygen consumption of tumour tissues increases, which further aggravates the hypoxia of tumour tissues. HIF-1α is a pivotal transcription factor involved in many biological processes, such as tumour cell survival, angiogenesis, invasion and tumour therapy. [11][12][13] It is reported that HIF-1α has been involved in the process of tissue fibrosis and cancer progression. 14 According to the literature, deletion of HIF-1α in FAP positive fibroblasts accelerates the growth of breast cancer in transgenic mice. 15 The loss of HIF-1α in endothelial cells restrains the growth of spontaneous breast tumour. 16 However, other studies suggest that HIF-1α induces the metabolic reprogramming of CAFs and increases glycolysis, thereby promoting tumour growth in breast cancer. 17 In pulmonary fibrosis, HIF-1α/PDK1-mediated glycolytic reprogramming can promote myofibroblast differentiation and fibrosis. 18 Therefore, the function of HIF-1α in CAFs is multifaceted and complex. Nevertheless, the role of HIF-1α in CAFs of LC has not been studied to date. As hypoxia is a common phenomenon of LC, it is very important to study the exact effect of HIF-1α on CAFs.
In the present study, we found that the HIF-1α-expressed fibroblasts promoted the growth of LC. Our results also showed that the expression of HIF-1α was increased under the stimulation of hypoxia, TGF-β1 and tumour cells CM, which were necessary for the activation of CAFs and LC growth. Finally, the mechanistic study revealed that HIF-1α-expressed fibroblasts accelerated the growth of LC through secreting CCL5.

| Tumour models
All mice were maintained under specific pathogen-free conditions and conducted with approval from the First Affiliated Hospital of Zhengzhou University Ethics Committee. The mouse spontaneous LC model (TetO-EGFR L858R ; CCSP-rtTA) was a gift of Professor Lin Xi from Tsing Hua University. To induce the formation of the lung tumours, the mice were treated as described previously. 19

| Real-time quantitative PCR
Total RNA was extracted using RNAiso plus (TAKARA, #1089527) and cDNA was synthesized using a prime script RT reagent kit with gDNA Eraser (TAKARA, RR047A). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using SYBR green™ primix Ex Taq™ Ⅱ(TAKARA, #RR820A) by Step One ® sequence detection system (Applied Biosystems). The relative RNA expression was calculated by using 2 −ΔΔCt method and the ribosomal protein GAPDH was used as a control gene to obtain normalized values. The indicated primers are shown in Table S1.

| Immunofluorescence assay
OCT-embedded tissues were sectioned at 6 μm thickness. The frozen slides or cells were fixed with 4.0% formaldehyde and incubated for 15 minutes. Cells were incubated with 0.1% TritonX-100 for 15 minutes. Then, the cells and frozen slides were blocked in PBS containing 3% bovine serum albumin for 30 minutes at 37℃. All of those patients were provided information about age, gender, pathological types and TNM stage. Each patient involved in this study was written informed consent, and the study was obtained with approval from the First Affiliated Hospital of Zhengzhou University Ethics Committee.

| Condition medium
iMEF cells and LLC cells were cultured in regular growth medium to 70% confluence. Then, cells were washed twice with PBS and cultured with fresh DMEM for 3 days. After that, the CM was centrifuged at 12,000 g for 5 minutes to remove cell debris and stored at −80℃. were co-injected into the mouse abdomen subcutaneously. Six days after injection, the tumour growth and weight were monitored every two days and the tumour volume was calculated as length × width × width/2. Eighteen days after injection, the mice were killed and the tumour tissues were embedded in OCT.

| Measurement of cell viability
Cells were cultured into 96-well plates and placed in the IncuCyte live-cell imaging system (Essen Bio Science). Cells were observed using phase microscopy with 10× objective (NiKon, #MRH00101) and calculated by IncuCyte analysis software. In addition, 50 cells were cultured into 6-well plates and cultured for 7 days. Then the cell clones were fixed with 4.0% formaldehyde for 15 minutes and stained with 0.1% purple crystal. The results of cell clones were detected by ChemiDoc MP Imaging System (Bio-Rad).

| Measurement of secreted cytokine
The concentration of CCL5 in fibroblasts CM were measured using a RANTES Mouse ELISA Kit (Invitrogen, KMC1031) according to the manufacturer's instructions.

| Data analysis
Data analysis was performed using the SPSS software version 17.0. All results were presented as mean ± SD. The significance of difference was assessed by t tests or variance analysis. The P values < 0.05, .01 and 0.001 were considered statistically significant.

| HIF-1α is highly expressed in CAFs of LC
HIF-1α is a transcription factor that is highly expressed in LC cells. 21 However, the role of HIF-1α in LC CAFs has not been fully investigated. To explore the relationship between the expression of HIF-1α and CAFs in LC, the expression level of HIF-1α was evaluated in the primary lung CAFs and NFs from LC patients. α-SMA is an activation marker of fibroblasts in the tumour microenvironment and inflammation environment, which usually reflects the location and activity of CAFs in cancer. 3 As shown in our results, the immunofluorescence analysis revealed that the fluorescence intensity of HIF-1α and α-SMA in human LC tissue was significantly higher than that in normal lung tissues ( Figure 1A). Next, to further study the expression of HIF-1α in CAFs, human CAFs (hCAFs) and NFs (hNFs) were isolated from lung tissues of LC patients F I G U R E 1 HIF-1α is highly expressed in CAFs of LC. A, Representative immunofluorescence images of HIF-1α (red) and α-SMA (green) in the frozen sections from human lung cancer tissues and normal lung tissues (Scale bar, 50 μm). B, The protein levels of COL1A2, FAP, α-SMA and HIF-1α were determined by western blot from hCAFs and their counterpart hNFs isolated from three lung cancer patients. C, The doxycycline-induced mouse spontaneous lung cancer model (TetO-EGFR L858R ; CCSP-rtTA) was established. D, Representative immunofluorescence images of HIF-1α (red) and α-SMA (green) in the frozen sections from mouse lung cancer tissues and normal lung tissues (Scale bar, 50 μm). E, The protein levels of COL1A2, FAP, α-SMA and HIF-1α were determined by western blot from mCAFs and mNFs isolated from mouse lung cancer tissues and lung normal tissues Doxycycline(2 mg/mL) in drinking water for 3 months and normal subjects according to the previously published protocol. 19 For the identification of the purity in the isolated hCAFs and hNFs, we detected the expression of well-recognized markers including α-SMA, COL1A2 and FAP, and as expected, were at higher levels in CAFs. Meanwhile, HIF-1α was up-regulated in hCAFs compared with hNFs ( Figure 1B). To eliminate individual human differences, a doxycycline-induced spontaneous LC model (TetO-EGFR L858R ; CCSP-rtTA) was established ( Figure 1C). Similar to human tissues, the co-staining intensity of α-SMA and HIF-1α in mouse LC tissues was higher than that in normal lung tissues ( Figure 1D). As α-SMA was a non-specific marker of fibroblasts, we then co-stained HIF-1α, α-SMA with ER-TR7 and found that HIF-1α is highly expressed on CAFs ( Figure S1A,D). We also isolated mouse CAFs (mCAFs) and mNFs (mNFs) from LC and normal lung tissues. We found that α-SMA, COL1A2, FAP and HIF-1α were highly expressed in mCAFs, which was consistent with the results observed in hCAFs and hNFs ( Figure 1E). To avoid the degradation of HIF-1α, we treated CAFs and NFs with MG-132 and found that HIF-1α was highly expressed in hCAFs and mCAFs ( Figure S1B,E).
We also detected the gene expression of HIF-1α in mCAFs and mNFs. Compared with NFs, the gene expressions of Hif1a were higher in CAFs of human lung cancer tissues and mouse lung cancer tissues ( Figure S1C,F). These findings support the notion that HIF-1α is highly expressed in lung CAFs.

| Up-regulation of HIF-1α induces the transformation of fibroblasts into CAFs
It is well documented that hypoxia up-regulates the expression of HIF-1α. 13 To gain further insights into the effects of HIF-1α on fibroblasts, primary MEF cells were isolated from C57 mouse embryo and treated with hypoxia. After 24 hours of treatment, a significant up-regulation of Hif1a was observed (about 5-fold) ( Figure 2A).
Simultaneously, hypoxia drastically facilitated the gene expression of Acta2, Pdpn, Fap and S100a4 ( Figure 2B), which were reported as CAFs markers. 3 After the treatment of hypoxia in MEF cells, we also found HIF-1α, α-SMA and COL1A2 increased in a time-dependent manner at the protein levels ( Figure 2C). These results suggest that hypoxia can up-regulate the expression of HIF-1α and induce fibroblasts to transform into CAFs.
Cocl 2 is used to mimic hypoxia to induce the expression of HIF-1α. 22 TGF-β1 and tumour cell CM are reported to activate the fibroblasts and increase the deposition of ECM. 8 To investigate the expression of HIF-1α during the activation of fibroblasts, MEF cells were treated with three kinds of stimulating factors (TGF-β1 and tumour cell CM, as well as Cocl 2 ). First, we confirmed that Hif1a expression was up-regulated at the RNA level ( Figure 2D). At the same time, we also detected the expression of HIF-1α at the protein level and found that the expression of HIF-1α was increased under the stimulators ( Figure 2E). To further determine the effect of HIF-1α expression on the activation of fibroblasts, we examined the effects of TGF-β1, tumour cell CM and Cocl 2 on Acta2 and Col1a2, which are markers of CAFs. 9 As shown in the figure, TGF-β1, tumour cell CM and Cocl 2 notably increased the expression of Col1a2 and Acta2 of fibroblasts at RNA levels ( Figure 2D). Moreover, the expression of α-SMA and COL1A2 was up-regulated, consisted with HIF-1α at protein levels ( Figure 2E)

| HIF-1α is essential for the activation of fibroblasts
In order to determine the relationship between HIF-1α and the activation of fibroblasts, we conducted a series of experiments in vitro. First, the MEF cells were treated with Cocl 2 and HIF-1α inhibitor (KC7F2) to investigate the effect of HIF-1α on the activation of fibroblasts. 23 As shown in Figure  Cocl2 ( Figure 3A). In addition, KC7F2 also could attenuate the effect of TGF-β1 and tumour cell CM on the expression of α-SMA and COL1A2 ( Figure 3B).
Furthermore, it was also observed that HIF-1α KO in fibroblasts restrained the gene expression of Acta2, Pdgfrα and Pdpn ( Figure 3C,D). Then, the HIF-1α MOCK fibroblasts and HIF-1α KO fibroblasts were treated with TGF-β1, tumour cell CM and Cocl2, respectively. The expressions of HIF-1α, α-SMA and COL1A2 were increased in HIF-1α MOCK fibroblasts, but did not alter in HIF-1α KO fibroblasts, suggesting that HIF-1α was involved in the activation of fibroblasts ( Figure 3E). We also detected the gene expression of Epas1 in HIF-1α MOCK and HIF-1α KO cells and found that the expression of Epas1 is decreased in HIF-1α KO cells ( Figure S2).
Overall, these data indicate that HIF-1α is necessary to mediate the activation of fibroblasts.

| Knockout of HIF-1α in fibroblasts attenuates the growth of LC
Cancer-associated fibroblasts are known to have an important role in tumour growth. 4 To identify whether fibroblasts with HIF-1α expression contribute to the promotion of tumour growth in vivo, we co-injected HIF-1α MOCK fibroblasts or HIF-1α KO fibroblasts with LLC cells into the C57 mice to detect the growth of lung tumour ( Figure 4A). As shown in our data, the tumour formation and growth in HIF-1α KO fibroblasts-injected group were significantly lower than that in HIF-1α MOCK fibroblasts-injected one, and the tumour volume and weight in the experimental mice were significantly smaller than those in the control group, while the mice weights had no obvious difference between two groups ( Figure 4B-D). Interestingly, the expression of CD31 in HIF-1α KO fibroblasts tumours is similar to that in HIF-1α MOCK fibroblasts ( Figure S3). PCNA and Ki-67 are the markers of cell proliferation. To explore the mechanism of the HIF-1α in fibroblasts promoting tumour growth, PCNA and Ki-67 immunofluorescence analysis were used. We found that PCNA and Ki-67 were abundant in HIF-1α MOCK fibroblasts tumour tissues ( Figure 4E). Those results show that the loss of HIF-1α in fibroblasts restrains the lung tumour growth by suppressing the tumour cell proliferation.

| HIF-1α-expressed fibroblasts secrete CCL5 to drive the growth of LC
To determine whether the HIF-1α-expressed fibroblasts contribute   Figure 5C), we speculated that CCL5 might play important roles in the promotion of tumour cell growth. We then detected the expression of CCR5 in LLC, MEF, A549 and MRC-5 cells. Our data showed that CCR5 was highly expressed in LLC and A549 LC cells ( Figure S4A). TCGA database showed that the expression of CCR5 was significantly increased in human LC ( Figure S4B). Next, ELISA analyses showed that HIF-1α MOCK fibroblasts secreted more CCL5 than HIF-1α KO fibroblasts  Figure 5D).
Furthermore, SC75741, a p-p65 inhibitor also inhibited the secretion of CCL5 in HIF-1α MOCK fibroblasts, rather than HIF-1α KO fibroblasts ( Figure 5E). Those results suggested that the HIF-1αexpressed fibroblasts secreted CCL5 via NF-κB signalling pathway, which further promotes the tumour growth.
In conclusion, our data indicated that HIF-1α was highly expressed in lung CAFs. Additionally, hypoxia, TGF-β1 and tumour cell CM up-regulated the expression of HIF-1α in fibroblasts, inducing the conversion of fibroblasts into CAFs. Moreover, HIF-1α-expressed CAFs secreted CCL5 by activating NF-κB signalling pathway, thus promoting the tumour growth of LC ( Figure 6).

| D ISCUSS I ON
Recently, many genetic engineering models have been developed to study LC. Several driver oncogenes are identified in human genome research, such as k-ras, EGFR mutant, which have been altered to induce LC models. 27 In this study, TetO-EGFR L858R ; CCSP-rtTA genetic In the previous study, α-SMA is used to mark the fibroblast. [28][29][30] Furthermore, α-SMA is significantly up-regulated after fibroblast activation and used as a surface marker for fibroblast subset. 31  can bind prolyl-hydroxylated HIF-1α and promote its proteasomal degradation. 34 To eliminate the degradation of HIF-1α, we treated CAFs and NFs with MG-132, the inhibitor of proteasome, and found that HIF-1α was indeed highly expressed in CAFs. Researchers found that three types of CAFs including myCAFs, iCAFs and apCAFs had been identified in pancreatic ductal adenocarcinoma. Among them, HIF-1α is significantly up-regulated in iCAFs, which is consistent with our data. 35 As the gene regulation is higher in our results and the proteasome degradation could not change the high expression of HIF-1α in CAFs, which results the expression of HIF-1α in CAFs is higher. However, it is unclear whether there are upstream genes in CAFs that regulate the expression of HIF-1α, it needs to further investigate for us.
Hypoxia is the main characteristic of LC microenvironment and also remodels the composition of TME through inadequate oxygen availability. 15 such as a-SMA and COL1A2. However, the relationship between HIF-1α expression and fibroblasts activation is not clear in LC.
CRISPR-cas9 was used to knock out the expression of HIF-1α. Due to the off-target effect of CRISPR-cas9 technology, the upstream molecules which regulate the expression of HIF-1α may be targeted.
That's why we choose H1-1 to conduct the following experiments.
HIF is a heterodimer consisting of three alpha subunits (HIF-1α, HIF-2α, and HIF-3α). 38 Under hypoxic conditions, both HIF-1α and HIF-2α are stabilized and the main function of HIF-2α is involved in regulating EPO and IRS2 in liver. 39 As shown in the results, when the expression of HIF-1α was knocked out, the expression of HIF-2α was decreased. Therefore, HIF-2α can't compensate the loss of HIF-1α in fibroblasts.
Fibroblasts are activated under the stimulation of tumour microenvironment, including TGF-β1, PDGF, SHH, BMP, IL-1, IL-6 and tumour necrosis factor (TNF). 3,5,7 The activation of fibroblasts in TME is not only by cytokines and the physical factors, but also by the changes of cells themselves. Among these, TGF-β1 signalling activates the resident fibroblasts to increase the metastatic potential and chemotherapy resistance of tumour cells. 40,41 Researchers also found that all subtypes of CRC with poor prognosis have common genetic programs induced by TGF-β1 in tumour stromal cells. 42 In addition to TGF-β1, tumour cell CM is commonly used to stimulate the fibroblasts. 43  hypoxia, but also can be regulated by oncogene activation or tumour suppressor gene inactivation. 45 We speculate that Igf1, Igf2 and Il6 may be up-regulated through other pathways after hypoxia stimulation, and that they are not the targets of HIF-1α. However, Ccl5 was greatly reduced in HIF-1α KO fibroblasts and mostly up-regulated in HIF-1α MOCK fibroblasts treated with hypoxia. Therefore, Ccl5 might be the target of HIF-1α in fibroblasts and was selected in the following experiment.
CCL5 mediates diverse biological functions and its main receptor is C-C chemokine receptor type 5 (CCR5). 46 The secretion of CCL5 mainly comes from T lymphocytes, macrophages, platelets, fibroblasts and several types of tumour cells, which is not only related to the immune response against tumours, but also to the cancer progression and metastasis. 47,48 In ovarian cancer, the increased miR-155 expression can transform fibroblasts into CAFs, thereby increasing the production of CCL5 and promoting the growth of tumour cells. 49 We found that the expression of CCR5 was highly expressed in LC cells. In summary, this study indicates that HIF-1α is highly expressed in CAFs, and HIF-1α-expressed fibroblasts secreted CCL5 by activating NF-κB signalling pathway, thus promoting the tumour growth of LC. Our study identify that HIF-1α is an essential factor of CAFs in LC and targeting HIF-1α-expressed CAFs has potential in future anticancer therapy.

ACK N OWLED G M ENTS
This work was supported by the National Natural Science Foundation of China (Grant Number: 81630068 and 82073231).

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data supporting the findings of this study are available within the article and its Supporting Information files.