Expression of HYOU1 via reciprocal crosstalk between NSCLC cells and HUVECs control cancer progression and chemoresistance in multicellular tumor spheroids


 Background

Among all cancer types, lung cancer ranks highest worldwide in terms of both incidence and mortality. The crosstalk between lung cancer cells and their tumor microenvironment (TME) has begun to emerge as the “Achilles heel” of the disease and thus constitutes an attractive target for anticancer therapy. We previously revealed that crosstalk between lung cancer cells and endothelial cells (ECs) induces chemoresistance in multicellular tumor spheroids (MCTSs).
Methods

In this study, we idientified that factors secreted in response to crosstalk between ECs and lung cancer cells play pivotal roles in the development of chemoresistance in lung cancer spheroids.
Results

We determined that the expression of hypoxia up-regulated protein 1 (HYOU1) in lung cancer spheroids was increased by factors secreted in response to crosstalk between ECs and lung cancer cells. Direct interaction between lung cancer cells and ECs also caused an elevation in the expression of HYOU1 in MCTSs. Inhibition of HYOU1 expression not only suppressed stemness and malignancy, but also facilitated apoptosis and chemosensitivity in lung cancer MCTSs. Inhibition of HYOU1 expression also significantly increased the expression of interferon signaling components in lung cancer cells. Moreover, the activation of the PI3K/AKT/mTOR pathway was involved in the HYOU1-induced aggression of lung cancer cells.
Conclusion

Taken together, our results identify HYOU1, which is induced in response to crosstalk between ECs and lung cancer cells within the TME, as a potential therapeutic target for combating the aggressive behavior of cancer cells.


Introduction
Lung cancer is one of most common cancer types and is the leading cause of cancer death worldwide.
Because lung cancer cells often exhibit abnormally high expression of epidermal growth factor receptor (EGFR) or mutations in the EGFR gene, EGFR inhibitors are currently used as the rst-line treatment for advanced lung cancer [1][2][3]. However, because treatment with EGFR inhibitors can lead to the development of mutations conferring resistance to such treatments (e.g., T790M), these drugs have limited therapeutic e cacy in lung cancer [4,5]. Anaplastic lymphoma kinase [6,7], ROS proto-oncogene1 [8,9], neurotrophic receptor tyrosine kinases [10][11][12] and the B-Raf proto-oncogene [13,14] have also emerged as possible therapeutic targets for lung cancer, but drugs based on these targets are less common, and further study of their side effects is still required. Hence, the development of targeted drugs has not yet signi cantly improved the prognoses of patients with lung cancer.
Recent studies have shown that lung tumor heterogeneity is a major cause of treatment failure due to chemoresistance [15,16]. In lung cancers, tumors develop concurrently with the tumor microenvironment (TME), which includes the vasculature, in ltrating immune cells, stromal broblasts, signaling molecules, and extracellular matrix surrounding the tumor [17], as evidenced by regions of aberrant angiogenesis, desmoplasia, acidosis, and hypoxia [18]. Within the TME, endothelial cells (ECs) have attracted attention as a key player in cancer progression due to their association with tumor cell angiogenesis, proliferation, and invasion, which have all been linked to the EC-mediated expression of various angiogenic factor receptors and remodeling of the extracellular matrix [19]. [19]. In our previous study, we elucidated that the direct interaction between lung cancer cells and ECs induces chemo-and radioresistance by facilitating the endothelial-to-mesenchymal transition (EndMT) in lung cancer cells [20][21][22].
Utilizing three-dimensional (3D) multicellular culture systems and modeling tumor interactions with stromal components are essential to establishing more clinically relevant tumor models [20,23,24]. Therefore, we used multicellular tumor spheroids (MCTSs) to identify factors that induce chemoresistance in response to crosstalk between lung cancer cells and ECs.
Here, we identi ed hypoxia upregulated protein 1 (HYOU1) as a factor secreted in response to crosstalk between ECs and lung cancer cells, and sought to elucidate its functional role in lung cancer chemoresistance and tumor growth. HYOU1, also known as oxygen-regulated protein 150 and glucoseregulated protein 170, is a member of the heat shock protein 70 family and play important roles in hypoxia/ischemia. HYOU1 is crucial for the processing and maturation of vascular endothelial growth factor A during angiogenesis. Extracellular HYOU1 from tumor cells acts as an immunomodulator in the TME [25][26][27][28], and expression of HYOU1 has been correlated with poor prognoses in patients with certain cancers, including breast cancer [29], nasopharyngeal carcinoma [30], renal cancer and thyroid cancer [31]. Further, HYOU1 upregulation has been linked to chemoresistance in various tumors [31][32][33]. However, the functions of HYOU1 in lung cancer remain largely unknown.
In this study, we investigated the effects of HYOU1 upregulation on the progression and chemoresistance of MCTSs. Based on our observations, we also discuss future therapeutic opportunities for lung cancer.

Materials And Methods
Cell line and cell culture NCI-H460 cells, A549 cells, H1299 cells, and PC9 cells were obtained from the Korean Cell Line Bank.

Generation of lung cancer spheroids with HUVECs
Lung cancer cells (NCI-H460 cells, or A549 cells) with or without HUVECs at a ratio of 5:5, or 7:3 were seeded at a density of 6 × 10 3 cells/well in 96-well round-bottomed ultra-low attachment microplates (Corning, NY, USA) with 80 µl of complete medium for 72 h. siHYOU1 transfection in lung cancer cells Lung cancer cells (NCI-H460 cells, A549 cells, or H1299 cells) were incubated in complete medium when the cells reached 60-70% con uence in 100-mm dish (Nunc, Thermo Scienti c, Waltham, MA, USA) at 37 °C incubator for 24 h. After washing for two times, 6 µM siRNA targeting HYOU1 (Dharmacon, Lafayette, CO, USA) and 40 pM lipofectamine RNAiMAX reagent (Invitrogen, Eugene, OR, USA) diluted in Opti-MEM (Gibco, Grand Island, NY, USA) medium were treated in fresh medium without supplements at 37 °C incubator for 24 h. After washing for two times, 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco, Grand Island, NY, USA) was treated for 3 min at 37 °C incubator, and then added equal amounts of complete medium. The cell suspensions were centrifuged at 1,300 rpm for 3 min, and then the cells were counted.

Cell death detection in spheroid
Non-speci c SiRNA (SiCont) or HYOU1 SiRNA (SiHYOU1) transfected for 24 h or 5, 10, 20, or 40 µM of ge tinib and cisplatin (all from Sigma-Aldrich, St. Louis, MO, USA) treated for 48 h, lung cancer cells (NCI-H460 cells, A549 cells, or H1299 cells) with or without HUVECs were seeded at a density of 6 × 10 3 cells/well in 96-well round-bottomed ultra-low attachment microplates (Corning, NY, USA). After 2 or 3 days, the spheroids cell death was detected using the cell-impermeant viability indicator ethidium homodimer-1 (EthD-1; Invitrogen, Eugene, OR, USA). EthD-1 is a high-a nity nucleic acid stain that uoresces weakly until bound to DNA, whereupon it emits red uorescence (excitation/ emission maxima 528/617 nm). Spheroids were incubated in 4 µM EthD-1 in complete medium for 30 min in a 37 °C incubator, and images were obtained and the intensity of EthD-1 uorescence measured using the Operetta® High Content Screening System (Perkin Elmer, Waltham, MA, USA). Fluorescent intensity analysis was performed using the Harmony software (Perkin Elmer, Waltham, MA, USA).

Microarray analysis
Global gene expression analysis was performed using Affymetrix GeneChip® Human Gene 2.0 ST Arrays.
Total RNA from non-speci c SiRNA (SiCont) or HYOU1 SiRNA (SiHYOU1) transfected NCI-H460 cells was isolated using the RNeasy Mini kit (Qiagen, Hilden, Germany). RNA quality was assessed using an Agilent 2100 Bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies), and the quantity was determined using a Nanodrop-1000 Spectrophotometer Thermo Scienti c). We used 300 µg of each RNA sample as input for the Affymetrix procedure, as recommended in the manufacturer's protocol (http:// www.affymetrix.com). Brie y, 300 ng of total RNA from each sample was converted to double-stranded cDNA using a random hexamer incorporating a T7 promoter, and ampli ed RNA (cRNA) was generated from the double-stranded cDNA template though an in vitro transcription (IVT) reaction and puri ed using the Affymetrix sample cleanup module. cDNA was regenerated through randomly primed reverse transcription using a dNTP mix containing dUTP. The cDNA was then fragmented by uracil-DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease (APE 1) restriction enzymes, and end-labeled via a terminal transferase reaction incorporating a biotinylated dideoxynucleotide. Fragmented endlabeled cDNA was hybridized to the GeneChip® Human Gene 2.0 ST array for 17 h at 45 °C and 60 rpm, as described in the Gene Chip Whole Transcript (WT) Sense Target Labeling Assay Manual (Affymetrix). After hybridization, the chips were stained and washed in a Genechip Fluidics Station 450 (Affymetrix) and scanned using a Genechip Array scanner 3000 7G (Affymetrix). The expression intensity data were extracted from the scanned images using Affymetrix Command Console software, version 1.1, and stored as CEL les.

Immunocytochemistry in lung cancer cells co-cultured with HUVECs spheroid
HUVECs were stained cell-labeling solution DiD (Molecular Probes, Eugene, Oregon, USA). DiD allows cell populations to be marked in distinctive uorescent colors for identi cation, whereupon it emits red uorescence (absorption/ emission maxima ~ 644/665 nm). HUVECs were incubated at a density of 1.

Colony formation assay
For colony formation assays, the lung cancer cells were transfected with SiCont or SiHYOU1. Then, the transfected cells were harvested and replaced at a density of 5 × 10 2 or 1 × 10 3 cells/6-well plate (Corning, NY, USA). The medium were changed every three days. Two weeks later, the colonies were washed with DPBS for two times and xed in 4% paraformaldehyde (Biosesang, Gyeonggi-do, Korea) for 24 h at 4 °C. After washing for two times, the colonies were stained with 0.5% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) in 20% methanol and washed until background appears light. Subsequently the colonies were photographed by an Eclipse TS100 microscope (Nikon, Tokyo, Japan) and counted.
Cell migration assay For cell migration assays, the lung cancer cells (NCI-H460 cells and A549 cells) were transfected with non-speci c SiRNA (SiCont) or HYOU1 SiRNA (SiHYOU1). Then, the treated cells were harvested and replaced at a density of 7 × 10 5 cells/6-well plate (Corning, NY, USA). After 1 day, the cells in the monolayer were vertically scratched using a 200 µl tip (Axygen, Corning, NY, USA) and incubated at 37 °C in a humidi ed atmosphere of 5% CO 2 . After incubation for 24 or 93 h, images of cell migration were obtained by an Eclipse TS100 microscope (Nikon, Tokyo, Japan).

Drug treatment in lung cancer cells
Lung cancer cells (NCI-H460 cells and H1299 cells) were seeded in complete medium when the cells reached 60-70% con uence in 6-well plate (Corning, NY, USA) at 37 °C in a humidi ed atmosphere of 5% CO 2 for. The cells were treated with 0.1 or 1 µM mTOR inhibitors, Torin2, WYE-125132, and PI3K inhibitors, GDC0032, PKI-402 (all from Selleckchem, Houston, TX, USA). After 1 day, cells were harvested.

Statistical analysis
All experiments were performed at least three times. The data are shown as the mean ± standard deviation (SD). A Student's t-test was used to assess statistically signi cant differences in excel. The signi cances were considered respectively with P values of P < 0.05, P < 0.01, and P < 0.001.

Crosstalk between ECs and lung cancer cells causes chemoresistance in lung cancer spheroids
We began by investigating whether interactions between ECs and lung cancer cells cause chemoresistance in lung cancer spheroids. First, we collected conditioned media (CM) from three different cell cultures: human umbilical vein endothelial cells (HUVECs/ECs), non-small cell lung cancer (NSCLC) cells (NCI-H460 or A549), and lung cancer cells co-cultured with HUVECs. We then added the three different CM to lung cancer spheroids in 3D cell culture conditions. We next compared the sensitivities of spheroids cultured in each medium to two anticancer drugs (ge tinib and cisplatin) [ Figure  1-A].
To detect cell death in the spheroids, we used the nucleic acid stain EthD-1, which emits uorescence upon binding the DNA of dead cells. Treatment with the EGFR inhibitor ge tinib signi cantly reduced cell survival in lung cancer spheroids grown in CM from cultured HUVECs or lung cancer cells, whereas spheroids grown in CM from lung cancer cells co-cultured with HUVECs showed strong resistance to ge tinib [ Figure 1-B].
We also evaluated the e cacy of cisplatin, a member of the platinum-based antineoplastic family of drugs, under the same conditions. Cisplatin also su ciently increased the intensity of EthD-1 staining in lung cancer spheroids grown in CM from cultured HUVECs or lung cancer cells. However, cisplatin did not alter cancer cell viability in spheroids grown in CM from lung cancer cells co-cultured with HUVECs [ Figure  1-C].
Taken together, these results suggest that factors secreted in response to crosstalk between ECs and lung cancer cells may play pivotal roles in chemoresistance in lung cancer spheroids.

HYOU1 expression is increased by factors secreted in response to crosstalk between ECs and lung cancer cells
We next sought to identify the factors inducing chemoresistance in lung cancer spheroids grown in CM from lung cancer cells co-cultured with HUVECs. To accomplish this, we subjected spheroids generated via the procedure shown in Fig. 1A to microarray analysis.
Analyses of the functional enrichment of genes with an absolute change of greater than 2-fold were performed using FunRich software. The microarray data revealed that 42 genes were signi cantly enriched and 103 genes were depleted in lung cancer spheroids grown in CM from lung cancer cells cocultured with HUVECs compared to spheroids grown in CM from lung cancer cells or HUVECs alone [ Figure 2-A, Supplementary Table I ].
Further, spheroids grown in CM from lung cancer cells co-cultured with HUVECs exhibited a greater than 1.5-fold increase in the expression of genes involved in gene expression, tRNA aminoacylation, and the unfolded protein response (UPR), whereas the expression of genes involved in the cell cycle and DNA replication were decreased [ Figure 2-B]. In terms of biological processes, we observed the enriched expression of genes involved in the protein metabolism and energy pathways, whereas we observed decreased expression of genes involved in cell communication and nucleic acid metabolism [ Figure 2-C].
Western blot analysis showed that aminoacyl-tRNA synthetase protein expression in lung cancer spheroids was not changed by factors secreted in response to crosstalk between ECs and lung cancer cells. As the PI3K/AKT signaling pathway plays a major role in cell proliferation, cell survival, and invasion in cancer, we next investigated the effects of HYOU1 on PI3K/AKT pathway components. HYOU1 protein expression and AKT phosphorylation at Ser473 were increased in H460 spheroids grown in CM from lung cancer cells co-cultured with HUVECs relative to H460 spheroids grown under normal conditions [ Figure 2-D]. This result prompted us to focus on functional roles of HYOU1 in TME of lung cancer.

Reciprocal crosstalk between NSCLC cells and HUVECs causes increased HYOU1 expression in MCTSs
We further sought to ascertain whether the direct interaction between ECs and lung cancer cells affects HYOU1 expression in MCTSs. To accomplish this, we co-cultured lung cancer spheroids with ECs and/or lung cancer cells (NCI-H460 or A549) in 3D cell culture. Spheroids co-cultured with both ECs and lung cancer cells showed enhanced compactness compared to spheroids cultured with lung cancer cells alone [ Figure 3-A]. Using the technique described in [ Figure 2D], we next estimated HYOU1 expression and PI3K/AKT signaling activation in these spheroids. We observed increased HYOU1 expression and AKT phosphorylation (Ser473) in spheroids co-cultured with ECs and lung cancer cells compared to spheroids cultured with lung cancer cells alone.
Because HYOU1 is a well-known component of the endoplasmic reticulum (ER) chaperone network, we also explored whether induction of HYOU1 is dependent on the canonical UPR pathway by inducing ER stress in spheroids co-cultured with ECs and lung cancer cells. Unexpectedly, we observed decreased expression of the three major ER stress sensor proteins [inositol-requiring enzyme-1 (IRE1), PKR-like ER kinase, and activating transcription factor-6 (ATF6)] in spheroids co-cultured with ECs and lung cancer cells [ Figure 3-B]. These results indicate that the increased HYOU1 expression observed in spheroids cocultured with ECs and lung cancer cells is unrelated to the ER stress response.
Next, we investigated which cells express more HYOU1 upon the co-culture of spheroids with ECs and lung cancer cells. To this end, we detected HYOU1 expression and localization via multilayer image acquisition using uorescence microscopy in lung cancer spheroids (NCI-H460 or A549) grown with HUVECs. Interestingly, HYOU1 expression was only observed in lung cancer spheroids and absent in HUVECs [ Figure 3-C].
Our previous study revealed that crosstalk between NSCLC cells and HUVECs induced strong chemoresistance in MCTSs [20]. In the present study, we investigated the effects of HYOU1 on the chemoresistance induced by the interaction between lung cancer cells and HUVECs in MCTSs. We generated tumor spheroids with suppressed HYOU1 expression by co-culturing HYOU1 siRNA-treated (siHYOU1) lung cancer cells with HUVECs, and treating the resulting spheroids with or without ge tinib or cisplatin. After MCTSs were allowed to form for 72 h, we assessed cell viability by measuring the levels of the apoptosis marker cleaved caspase-3. We observed that the suppression of HYOU1 expression in lung cancer cells caused an increase in the expression of cleaved caspase-3 in MCTSs [ Figure 3-D]. These results show that depletion of HYOU1 in lung cancer cells affects the drug sensitivity of the TME.

Depletion of HYOU1 inhibits tumor growth, and the stemness and expression of EMT-related proteins in lung cancer cells
Because HYOU1 is an ER-associated chaperone induced by hypoxia [34][35][36], we investigated its expression in lung cancer spheroids, which exhibit hypoxia. Spheroid (3D) cultures derived from various lung cancer cells exhibited increased expression of HYOU1 relative to monolayer (2D) cultures [ Figure 4-A].
According to analysis of data from The Cancer Genome Atlas program, HYOU1 is an unfavorable prognostic marker in renal and thyroid cancer, but is not prognostic in lung cancer. HYOU1 has a low speci city for expression in the lung cancer TME. Nevertheless, we examined whether HYOU1 controls lung cell growth to investigate the potential effects of altered HYOU1 expression on lung growth. Clonogenic survival was diminished by depletion of HYOU1 in H460 cells (40%), A549 cells (64.2%), and H1299 cells (46.2%) [ Figure 4-B].
To elucidate the functional roles of HYOU1, we next examined whether HYOU1 controls tumor growth and metastasis in a spheroid model similar to the lung cancer TME.
The siRNA-mediated depletion of HYOU1 signi cantly increased cell death in H460, A549, and H1299 spheroids [ Figure 4-C]. Expression of two apoptosis markers, cleaved PARP and cleaved caspase-3, were measured in H460 and A549 spheroids following HYOU1 depletion. The p38 MAPK is related to hypoxiainduced apoptosis [37,38] and ERK/MAPK is involved in resistance to apoptosis under hypoxic conditions [39,40]. Inhibition of HYOU1 expression induced p38 activation, but attenuated ERK activation, in H460 and A549 spheroids. However, suppressing HYOU1 expression did not alter the activation of p53 or HIF1 [ Figure 4-D].
To identify the possible effects of HYOU1 on cancer stem cell (CSC) populations of lung cancer cells, HYOU1-depleted lung cancer cells were cultured under spheroid-forming conditions, and the resulting spheroid number and size were analyzed [ Figure 5-A]. Inhibition of HYOU1 signi cantly attenuated the spheroid-forming capacity of lung cancer cells. As CD133 expression plays a critical role in the maintenance of stem-like properties in lung cancer [41,42], depletion of HYOU1 decreased the expression of CD133 in lung cancer cells [ Figure 5-B]. These results show that HYOU1 may also be involved in the propagation of CSCs in lung cancer.
Because CSCs have been associated with tumor initiation, therapeutic resistance, and metastasis, we next sought to determine whether inhibition of HYOU1 expression sensitizes lung cancer cells to anticancer therapies and thus enhances their e cacy. After lung cancer spheroids (NCI-H460 or A549) with or without siHYOU1 were subjected to ge tinib treatment, the intensity of EthD-1 staining in spheroids was measured. Depletion of HYOU1 expression markedly enhanced lung cancer spheroid chemosensitivity to ge tinib [ Figure 5-C]. To investigate the effects of HYOU1 on cell migration, we also performed wound healing assays following HYOU1 depletion in lung cancer cells and found that lung cancer cell migration was attenuated by HYOU1 depletion [ Figure 5-D]. We next measured the expression of epithelial-to-mesenchymal transition (EMT)-related proteins in HYOU1-depleted lung cancer cells. Expression of N-cadherin, α-SMA, vimentin, and collagen I were all decreased in HYOU1-depleted lung cancer cells [ Figure 5-E]. These results demonstrate that HYOU1 plays a pivotal role not only in inhibition of tumor growth and stemness but also in enhancing the anticancer e cacy of the lung cancer TME.

HYOU1 controls tumor growth via the alteration of interferon signaling in lung cancer cells
To reveal the molecular mechanism by which HYOU1 affects lung tumor growth, we performed gene expression pro ling on the HYOU1-depletion system in H460 cells. Using a fold difference cutoff of greater than 2.5-fold, we identi ed 44 genes that were differentially expressed between HYOU1-depleted Table II, III]. According to the Reactome Pathway Database, HYOU1 is functionally involved in the UPR, chromatin organization, and interferon (IFN) signaling [ Figure 6-A]. It is noticeable that expression of IFN-type I (IFN-α, β) were more increased than IFN-type II (IFN-γ) by depletion of HYOU1 expression. IFN-α, β have been found to be effective in reducing the growth of various tumor [41]. Western analysis also showed that expression of IFN-type I (IFN-α, β) were increased by inhibition of HYOU1 expression during process of cell death in lung cancer cells [ Figure  6-B]. HYOU1 expression is downregulated by the inhibition of the PI3K/AKT/mTOR pathway Although HYOU1 is a larger protein than GRP78, its overall structure is highly homologous to that of GRP78. Because the inhibition of the PI3K/AKT/mTOR signaling pathway suppresses GRP78 expression [42,43], we evaluated the effect of activating the PI3K/AKT/mTOR pathway on HYOU1 expression. Treatment with the mTOR inhibitors Torin2 and WYE-132, and the potent PI3K inhibitors GDC-0032 and PKI-402, signi cantly inhibited HYOU1 expression in H460 and H1299 cells [ Figure 7-A].

H460 cells and normal H460 cells [Supplementary
Because mTOR inhibitors could decrease an HYOU1 expression in H460 and H1299 cells, we next examined whether mTOR controls HYOU1 expression by using siRNAs for mTOR and HYOU1 in H460 and H1299 cells. Western blot analysis revealed that siRNA against mTOR e ciently depleted HYOU1 expression, whereas the inhibition of HYOU1 did not affect mTOR expression [ Figure 7-B] and H1299 cells. These resutls suggest that mTOR could regulate the expression of HYOU1 in lung cancers.

Discussion
ECs are the most frequently studied components of the TME. Generally, ECs undergo a phenotypic transformation to activated myo broblast-like cells through the EndMT [20]. Cancer cells stimulate the activation of the EndMT in ECs, and transformed ECs support cancer progression by secreting diverse cytokines, growth factors, and proteins of the ER membrane protein complex. Secretomics-the analysis of the secretome (all the secreted proteins of a cell, tissue, or organism)-is important to the discovery of cancer biomarkers because secreted proteins facilitate communication between distinct cells in multicellular organisms and control a broad range of physiological functions [44]. Because CM obtained from the co-culture of lung cancer cells and HUVECs promoted robust chemoresistance in lung cancer spheroids [ Figure 1], we ascertained that the paracrine effects of ECs on lung cancer chemoresistance should be further investigated in 3D culture conditions to identify the chemoresistance-inducing factor(s) present in the co-cultured CM.
Expression of many genes, especially genes related to tRNA aminoacylation and the UPR, was highly increased in lung cancer spheroids following treatment with CM from co-cultured lung cancer cells and Table I]. In the present study, we focused speci cally on the functional roles of HYOU1 in lung cancer cells.

HUVECs [Supplementary
HYOU1, which is a well-characterized ER chaperone and the largest glucose-regulated protein, has an ADP-ATP exchange function via its interaction with GFP78 [45], enabling it to protect cancer cells from cell death by delaying the onset of the UPR and binding to ER stress sensors. HYOU1 has also been linked to cancer development and progression through its facilitation of chemoresistance, tumor invasion, and angiogenesis in various tumor types. Conversely, HYOU1 has been shown to have potent antitumor effects in vivo dependent on cytotoxic CD8 + T cells, so it may be an effective target in a new vaccine platform to generate a therapeutic antitumor response [26]. Given these con icting data, the functions of HYOU1 remain controversial and must be studied in the TME.
In our study, hypoxia increased the expression of HIF1 and HYOU1 in lung tumor spheroids, whereas silencing of hypoxia-induced HYOU1 suppressed tumor growth [ Figure 4]. CSC populations can cause tumor recurrence, metastasis, and treatment failure in patients with lung cancer. Hypoxia stimulates the propagation of CSC populations and triggers increased expression of HYOU1 [46]. In the present study, depletion of HYOU1 expression not only suppressed the propagation of CSCs via the inhibition of CD133 expression but also regulated metastasis and chemoresistance in lung cancer cells [ Figure 5]. Hence, we consider HYOU1 to be an attractive target for lung cancer therapeutics. IFN-α, β induced apoptotic cell death in various tumors and also in triggering the anti-tumor immune response in humans [41,[47][48][49][50]. In lung cancer cells, inhibition of HYOU1 increased the expression IFN-α and IFN-β during cell death [ Figure 6]. Although GRPs are mainly induced in response to ER stress, induction of HYOU1 in spheroids co-cultured with ECs and NSCLC cells was not dependent on the ER stress response [ Figure 3B]. Because GRPs are also mediated by PI3K/AKT/mTOR signaling [42,51,52], we tested the effects of mTOR and PI3K inhibitors on the expression of HYOU1 in lung cancer cells.
Increases in both HYOU expression and pAKT (Ser473)/mTOR occurred simultaneously in H460 spheroids grown in CM from lung cancer cells co-cultured with ECs and in MCTSs co-cultured with ECs and NSCLC cells [ Figure 2-D, 3-B]. We showed that induction of HYOU1 via the activation of PI3K/AKT/mTOR signaling facilitates tumor malignancy in lung cancer. HYOU1 also promotes cell growth and metastasis by modulating the PI3K/AKT pathway in epithelial ovarian cancer [53]. Several mTOR-targeted agents are under clinical development for the treatment of lung cancer [54].

Conclusions
In this study, we sought to elucidate the mechanisms underlying HYOU1-induced chemoresistance in MCTSs co-cultured with ECs and NSCLC cells. Our study demonstrated that depletion of HYOU1 suppresses tumor growth, chemoresistance, and migration via inhibiting CSC populations by increasing the expression of IFN-α and IFN-β in lung cancer cells. Expression of HYOU1 is modulated by the activation of the PI3K/AKT/mTOR pathway [ Figure 8]. Hence, selective inhibitors of HYOU1 expression could represent promising therapeutic targets for overcoming chemoresistance and tumorigenesis in lung cancer.

Consent for publication
All authors read and approved the nal manuscript for publication.

Availability of data and materials
Information is included in the Methods section.

Competing interests
The authors declare that they have no competing interests. Experimental schematic of CM obtained from cultured HUVECs (ECs), lung cancer cells (NCI-H460 or A549), and lung cancer cells co-cultured with HUVECs. The cells were cultured for 3 days in 2D culture conditions using the same number of cells and the same amount of media. Next, three different CM were added to lung cancer cells in 3D culture conditions for 2 days. The spheroids were then treated with drugs for 2 days, and EthD-1 staining was used to identify dead cells. (B and C) Immuno uorescence images of lung cancer spheroids grown in CM obtained from cultured HUVECs, cultured lung cancer cells, or lung cancer cells co-cultured with HUVECs. The cells were treated with 5, 10, 20, or 40 µM ge tinib (B) or cisplatin (C) for 2 days, and then stained with 4 µM EthD-1. The images were obtained using the Operetta® High Content Screening System, and the intensity of EthD-1 staining in lung cancer spheroids relative to controls was analyzed using Harmony software. The data shown are the means ± SD from three independent experiments; *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the control group.    Immuno uorescence and bright-eld images of lung cancer spheroids (NCI-H460, A549, and H1299) transfected with nonspeci c siRNA (SiCont) or HYOU1 siRNA (SiHYOU1). The spheroids were stained with 4 µM EthD-1. The images were obtained using the Operetta® High Content Screening System, and the intensity of EthD-1 staining in lung cancer spheroids relative to controls was analyzed using Harmony software. (D) Expression of cleaved caspase-3, cleaved PARP, HIF1, HYOU1, pp38, p53, and pErk1/2 in lung cancer spheroids (NCI-H460 and A549) transfected with nonspeci c siRNA (SiCont) or HYOU1 siRNA (SiHYOU1). The data shown are the means ± SD from three independent experiments; *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the control group.