PRMT5-mediated methylation of STAT3 is required for lung cancer stem cell maintenance and tumour growth

doi:10.21203/rs.3.rs-3113607/v1

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PRMT5-mediated methylation of STAT3 is required for lung cancer stem cell maintenance and tumour growth

https://doi.org/10.21203/rs.3.rs-3113607/v1

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published 17 May, 2024

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STAT3 is constitutively activated in many cancer types, including lung cancer, and can induce cancer cell proliferation and cancer stem cell (CSC) maintenance. STAT3 is activated by tyrosine kinases, such as JAK and SRC, but the mechanism by which STAT3 maintains its activated state in cancer cells remains unclear. Here, we show that PRMT5 directly methylates STAT3 and enhances its activated tyrosine phosphorylation in non-small cell lung cancer (NSCLC) cells. PRMT5 expression is also induced by STAT3, suggesting the presence of a positive feedback loop in cancer cells. Furthermore, methylation of STAT3 at arginine 609 by PRMT5 is important for its transcriptional activity and support of tumour growth and CSC maintenance. Indeed, NSCLC cells expressing the arginine 609 STAT3 mutant show significantly impaired tumour growth in nude mice. Overall, our study reveals a mechanism by which STAT3 remains activated in NSCLC and provides a new target for cancer therapeutic approaches.

The signal transducer and activator of transcription (STAT) protein family members were originally identified as regulatory transcription factors involved in the cellular response to interferons (IFNs)¹. Subsequently, STATs were found to be activated by various cytokines, cell growth factors, and hormones via the intracellular, non-receptor tyrosine kinases, Janus kinases (JAKs), to regulate cellular responses to these ligands^{1, 2}. STAT family proteins include STAT1–4, 5A, 5B, and 6. After being phosphorylated at tyrosine residues by JAKs, STATs can dimerize and translocate into the nucleus to regulate target gene expression in response to extracellular signals and control various cellular responses, such as immune system activities and cell growth^{3, 4, 5}. In cancer cells, STAT proteins are activated by increased expression of cytokine/growth factors and by aberrant activation of their signalling molecules. STATs are involved in tumour cell growth, resisting cell death, and cancer stem cell (CSC) maintenance via expression of their target genes^{3, 5}. The involvement of STAT family proteins in tumourigenesis and cancer progression has been widely reported, particularly STAT3 and STAT5, which are constitutively activated in many cancer cells and have been experimentally shown to be important in cancer progression^{5, 6}.

STAT3 was identified as a transcription factor that binds to interleukin-6 (IL-6)-responsive elements in the promoters of acute phase protein genes⁷. The IL-6/JAK/STAT3 signalling pathway plays a pro-inflammatory role by promoting the epithelial-mesenchymal transition process and many cellular responses, including immune responses, angiogenesis, and cell growth and differentiation^{4, 8, 9}. Furthermore, STAT3 is activated by various inflammation-related and immune regulatory cytokines and cell growth factors that have significant functions in wound healing and tissue repair. Therefore, excessive activation of STAT3 in cancer cells, as well as in cells in the inflammatory tumour microenvironment (TME), can cause an inflammation-driven repair response¹⁰. This is the so called "wounds that do not heal" model of cancer that can support tumour promotion and progression^{11, 12}. In cultured cells, a constitutively activated STAT3 mutant was shown to function as an oncogene¹³, suggesting that STAT3 itself can reprogram normal cells into CSCs^{14, 15}. In this context, STAT3 can help generate CSCs by inducing the expression of reprogramming factors, such as SOX2 and OCT4¹⁶. Moreover, the inflammatory TME may promote the establishment and maintenance of CSCs via STAT3^{17, 18}. In addition to these functions, STAT3 can induce metabolic reprogramming, a hallmark of cancer, by regulating various metabolic processes in cancer cells, including aerobic glycolysis, oxidative phosphorylation, glutamine metabolism, lipid synthesis, and lipid catabolism³. Therefore, inhibition of STAT3 activity is expected to inhibit cancer cell proliferation and suppress CSCs, but clinically effective drugs are not yet available¹⁰.

STAT3 activation is initiated by phosphorylation of the tyrosine residue (Y705) by JAK family kinases, JAK1, JAK2, JAK3, and TYK2. This tyrosine residue can also be phosphorylated by non-receptor kinases, such as SRC and ABL^{2, 4}. Tyrosine-phosphorylated STAT3 forms dimers that translocate to the nucleus to activate the expression of target genes. STAT3 is also phosphorylated at the serine 727 (S727) residue by mitogen-activated protein kinase (MAPK) protein family members, such as extracellular signal-regulated kinase (ERK) 1, ERK2, p38, and c-Jun N-terminal kinase (JNK)^{19, 20}. Mutant STAT3-expressing cells in which S727 was replaced with alanine did not affect the DNA-binding activity of STAT3, but decreased the transcriptional activity, suggesting that this phosphorylation is required for maximum STAT3 action. Moreover, expressing this mutant STAT3 in mice prevented the lethality associated with STAT3 deficiency, but its transcriptional activity was reduced by half ²¹. Additionally, acetylation and methylation of STAT3 can reportedly enhance its transcriptional activity^{19, 20}.

Constitutive activation of STAT3 has been reported in many cancer types. However, STAT3 gain-of-function mutations have been observed in some leukaemias, but less frequently in solid tumours²². Because STAT3 can induce the expression of cytokines/growth factors, and their receptors activate STAT3, it is possible that STAT3 is permanently activated by a positive feedback loop in the TME⁵. In JAK/STAT regulation systems, activation of STAT3 by JAK is inhibited by suppressor of cytokine signalling 3 (SOCS3), which is transcriptionally induced by STAT3²³. Thus, given the importance of STAT3 for cancer cell survival^{5, 24}, the mechanism of constitutive STAT3 activation in cancer cells needs to be fully elucidated. In the present study, we found that protein arginine methyltransferase 5 (PRMT5)-mediated methylation is important for constitutive activation of STAT3, as well as for CSC maintenance and tumourigenesis in lung cancer cells. These results suggest that targeting the arginine methylation of STAT3 by PRMT5 is a potential new therapeutic approach for treating lung cancer.

PRMT5 mRNA expression levels are upregulated in lung cancer cells. Lung cancer is a leading cause of cancer-related deaths, with more than 85% of lung cancer cases classified as non-small cell lung cancer (NSCLC)²⁵. The most common genetic driver alteration in NSCLC is mutations of epidermal growth factor receptor (EGFR) that result in constitutive activation. STAT3 is one of the key signalling mediators downstream of EGFR, and STAT3 is indeed constitutively activated in about 50% of NSCLC tissues and cell lines^{26, 27, 28}. In addition to this pathway, the HEDGEHOG (HH) pathway is known to be activated in NSCLC²⁹. Experimental evidence suggests that STAT3 activation is enhanced downstream of the HH signal³⁰. The HH signalling pathway plays a central role in the growth, patterning, and morphogenesis of organisms, and is also involved in adult organ homeostasis, stem cell maintenance, and cancer development^{31, 32, 33}. We previously found that HH signalling in cancer cells can induce expression of PRMT5, with arginine methylation by PRMT5 being important for activation of the effector transcription factor GLI1 protein³⁴. PRMTs are enzymes that can methylate arginine residues in certain proteins and have been reported to regulate gene expression, signal transduction, and mRNA splicing fidelity³⁵. Of the nine members of the PRMT protein family, PRMT5 is believed to be involved in cancer through various functions, including tumour growth³⁶, stem cell reprogramming and maintenance³⁷, and immune evasion³⁸. Furthermore, overexpression of PRMT5 has been observed in a variety of cancer types³⁹. Therefore, we first analysed PRMT5 expression levels in NSCLC samples. As shown in Fig. 1a/b, PRMT5 mRNA expression levels were enhanced in the two most common NSCLC subtypes, lung adenocarcinoma and lung squamous cell carcinoma, as determined by RNA sequencing (RNA-seq) transcriptomic data from The Cancer Genome Atlas (TCGA) database⁴⁰. PRMT5 expression levels and copy number were correlated, with higher PRMT5 expression being associated with worse overall survival in lung adenocarcinoma patients (Supplementary Fig. 1a, b). Moreover, mRNA expression of methylosome protein 50 (MEP50), an adaptor molecule between PRMT5 and its substrates^{41, 42}, was also enhanced (Supplementary Fig. 1c, d). This suggests that the functions of methylosomes containing PRMT5 are increased in NSCLC. PRMT5 expression levels were higher in NSCLC compared with other well-known HH signalling-enhanced cancers, such as esophagogastric, colorectal, prostate, and pancreatic cancers^{43, 44, 45, 46} (Fig. 1c). Ranking of PRMT5 expression in cancer cell lines with the Cancer Cell Line Encyclopedia (https://sites.broadinstitute.org/ccle/) also showed high PRMT5 expression levels in NSCLC cells (Fig. 1d). These results suggest that methylation by PRMT5 plays an important role in NSCLC, including cancer progression.

STAT3 activates PRMT5 gene transcription. Because PRMT5 mRNA expression levels were upregulated in NSCLC samples, we next analysed the relationship between the transcription factor STAT3, which is constitutively activated in NSCLC, and PRMT5 expression. Using RNA interference (RNAi) to knock down STAT3 expression (siSTAT3), we observed reduced PRMT5 protein expression levels in normally growing murine diploid NIH3T3 cells. STAT3 expression was induced by v-SRC, a typical oncogenic signalling molecule known to activate STAT3⁴⁷ (Fig. 2a), suggesting that STAT3 is involved in the regulation of PRMT5 expression in cancer cells. Moreover, PRMT5 mRNA expression was upregulated by STAT3 activator IL-6, while this induction was suppressed by STAT3 knockdown (Fig. 2b). EGF-induced PRMT5 mRNA expression was also inhibited by STAT3 inhibitor LLL-12 (Fig. 2c), suggesting that PRMT5 gene transcription is activated by STAT3. Indeed, the PRMT5 promoter, which includes putative STAT3 binding sites⁴⁸, was activated by a constitutively active mutant of STAT3¹³ (Fig. 2d–f) and STAT3 protein directly bound with the PRMT5 promoter region (Fig. 2g, h). Binding of STAT3 to the PRMT5 promoter was also confirmed in the UCSC Genome Browser database (Fig. 2i). Finally, STAT3-dependent constitutive expression of PRMT5 was also observed in the NSCLC cell lines HCC827 (EGFR mutant, adenocarcinoma; Fig. 2j) and A549 (EGFR wild-type, adenocarcinoma; Fig. 2k).

PRMT5 induces tumour growth and CSC maintenance in NSCLC cells. We next analysed the role of PRMT5 in HCC827 and A549 NSCLC cells. The proliferation rates of cells with knockdown of PRMT5 (Fig. 3a) and its adaptor molecule MEP50 (Fig. 3b) were slightly attenuated compared with that of the parental cells (Fig. 3c, d). Cancer arises from CSCs, also known as cancer-initiating cells, which have tumourigenic potential, self-renewal properties, and long-term tumour repopulating activity^{14, 49, 50}. It has been shown that CSCs can form spheres under low attachment culture conditions in media containing growth factors^{51, 52}. Indeed, these cells formed primary spheres and secondary spheres, characteristic of stem cells⁵³, but PRMT5 knockdown reduced their number (Fig. 3e–h). Certain reprogramming factors, such as SOX2 and OCT4, have induced pluripotent stem cell (iPSC)-inducing activity and are a hallmark of CSCs¹⁵. In sphere cells, the expression levels of these reprogramming factors were elevated (Supplementary Fig. 2). In addition, the population of highly activated ALDH cells, which is a hallmark of lung CSCs, was reduced in PRMT5 knockdown cells (Fig. 3i, j). These results suggest that PRMT5-mediated methylation is important for CSC maintenance. Like the decreased number of sphere-forming cells, the growth of A549 cell tumours transplanted in nude mice were also reduced in PRMT5 knockdown cells (Fig. 3k, l). Furthermore, this tumour growth inhibitory effect not only seen by constitutive knockdown, but also by inducible knockdown of PRMT5 and MEP50 (Fig. 3m, n).

STAT3 activity is upregulated by PRMT5-mediated methylation. Next, we analysed the functional effects of the PRMT5 and STAT3 interaction in cancer cells. The number of sphere-forming cells in HCC827 and A549 cells was increased by IL-6 stimulation, but this increase did not occur in PRMT5 knockdown cells (Fig. 4a, b). Related to this, the induction of MCP-1 mRNA, which is known to be regulated by the IL-6-STAT3 pathway⁵⁴, was suppressed by PRMT5 knockdown (Supplementary Fig. 3a, b). Furthermore, PRMT5 knockdown also suppressed the increase of sphere-forming cell number following hepatocyte growth factor (HGF) treatment, which is known to induce oncogenic transformation via the MET-STAT3 pathway⁵⁵ (Supplementary Fig. 3c, d). These results suggest that STAT3 is possibly involved in CSC maintenance downstream of PRMT5. Indeed, IL-6-induced Y705 phosphorylation was suppressed in PRMT5 knockdown cells (Fig. 4c), as was STAT3 transcriptional activity (Fig. 4d, e). However, PRMT5 knockdown did not affect the transcriptional activity of transcription factors NF-kB and HIF1⁵⁶, which are known to be activated in other cancer types (Supplementary Fig. 3e, f). In A549 cells, we found that PRMT5 was bound with STAT3 (Fig. 4f). Furthermore, STAT3 itself was arginine methylated in this complex, and its methylation was inhibited by the PRMT5 inhibitor (Fig. 4f). Formation of this complex with STAT3 and PRMT5 was suppressed by MEP50 knockdown (Fig. 4g), suggesting that STAT3 is methylated in the methylosome complex formed by PRMT5/MEP50^{41, 42}. In addition, PRMT5 was mainly located in the cytoplasm, implying that STAT3 methylation occurs there (Supplementary Fig. 3g, h). Previously, it has been shown that STAT3 plays an important role in v-SRC transformation, as dominant negative STAT3 mutants can inhibit v-SRC transformation⁵⁷. As shown in Supplementary Fig. 4a–f, tumour suppressor p53-deficient embryonic fibroblasts expressing v-SRC showed foci formation⁵⁸, a hallmark of cancer cells. However, this ability was reduced by PRMT5, MEP50, or STAT3 knockdown, suggesting that the PRMT5/MEP50/STAT3 pathway is important for CSC generation.

PRMT5-mediated STAT3 methylation is required for tumourigenesis and CSC maintenance in NSCLC cells. Candidate arginine methylation sites in STAT3 were predicted by PRmePRed (Fig. 5a, b). Then, wild-type STAT3 and STAT3 mutants were transiently expressed in STAT3 knockout BEAS2B cells (Supplementary Fig. 5). Amino acid substitution (arginine to lysine) at candidate methylation sites revealed that arginine 609 (R609K) was clearly methylated and arginine 518 (R518K) was weakly methylated by PRMT5. Correlated with the decrease in methylation by the arginine mutant, phosphorylation of STAT3 Y705 was also decreased (Fig. 5c). Furthermore, R609K mutant STAT3 lost most of its transcriptional activity in A549 cells (Fig. 5d), and stably expressing STAT3-R609K could also suppress Y705 phosphorylation in STAT3 knockout HCC827 cells (Fig. 5e).

Finally, we examined the impact of STAT3-R609K on tumour growth and CSC maintenance. STAT3 knockdown HCC827 cells showed slightly decreased cell proliferation rates, but these were increased to levels similar to the parental cells after wild-type STAT3 was expressed. However, STAT3-R609K mutant-expressing cells did not show recovered proliferation rates (Fig. 5f). Moreover, STAT3 knockdown cells had a markedly decreased number of sphere-forming cells, and the R609K mutant showed a reduced recovery of sphere-forming cells compared with wild-type STAT3 (Fig. 5g). Furthermore, tumour growth in nude mice was markedly reduced in STAT3 knockdown cells and was restored by wild-type STAT3 expression, similar to the parental cells, while this recovery was not observed in mutant STAT3-R609K-expressing cells (Fig. 5h–j). These results suggest that methylation of STAT3 R609 by PRMT5 is important for CSC maintenance and tumour growth in NSCLC.

This study reveals that PRMT5 can methylate and activate STAT3, which can then induce PRMT5 expression, thereby maintaining both in an activated state (Fig. 5j). These results reveal a previously unknown mechanism of STAT3 and PRMT5 activation. Furthermore, we found that activation of this PRMT5-STAT3 pathway is important for tumour growth and CSC maintenance in NSCLC. The molecular mechanism of the reprogramming process, in which normally differentiated cells are transformed into CSCs, is thought to involve similar regulators and control mechanisms as those of iPSCs¹⁵. Indeed, the reprogramming transcription factors and chromatin modifiers required for iPSC establishment have been shown to be associated with cancer development^{15, 59}. Because cancer is caused by stepwise changes to oncogene and tumour suppressor gene expression and/or protein activity^{60, 61}, cancer-promoting signals occurring as a result of these changes can induce cell reprogramming. In cultured cells, constitutively activated STAT3 mutant has been shown to function as an oncogene¹³, suggesting that STAT3 itself can reprogram normal cells into CSCs^{14, 15}. Indeed, it has been shown that STAT3 is involved in CSC generation by inducing the expression of reprogramming factors such as SOX2 and OCT4¹⁶. In the present study, we found that STAT3 knockdown could suppress fibroblast transformation by v-SRC and reduce the number of CSCs in NSCLC. These results suggest that STAT3 is important for CSC generation and maintenance, especially in NSCLC tumours. Thus, the mechanism of constitutive STAT3 activation may be important for CSC development, dedifferentiation of differentiated cancer cells into CSCs, and CSC maintenance.

In this study, we found that PRMT5 can activate STAT3 via arginine methylation. In previous work, PRMT5 knockdown was shown to suppress cell proliferation, while overexpression of PRMT5 could cause increased cancer cell proliferation^{36, 62}, suggesting a role in cancer cell growth. Moreover, PRMT5 is also important for maintaining pluripotency of mouse embryonic stem cells⁶³. Additionally, the induction of iPSCs by Yamanaka factors (OCT4, SOX2, KLF4, and C-MYC) is reduced in cells lacking PRMT5⁶⁴. Thus, PRMT5 is likely important for CSC generation and maintenance. We have previously shown that GLI1, the effector transcription factor in the HEDGEHOG pathway that is thought to be important in CSC development, is activated by PRMT5/MEP50³⁴. It has been proposed that HH signalling maintains the stemness of CSCs^{65, 66}, suggesting that PRMT5/MEP50 are also important for CSC maintenance. In the present study, we also found that methylation of STAT3 by PRMT5/MEP50 in NSCLC cells is important for its transcriptional activity, for the generation and maintenance of CSCs, and for tumour growth in mice. Therefore, although the dependence may vary by cancer type, our data demonstrate that the PRMT5/MEP50/STAT3 pathway is important for CSCs in NSCLC.

Activation of STAT3 by JAK leads to transcriptional induction of the JAK inhibitor SOCS3 by STAT3²³, suggesting that STAT3 activity can oscillate in JAK-activated cancer cells and EGFR-mutated cancer cells⁶⁷. Thus, in cancer cells that are dependent on STAT3 for survival, continuous activation of JAK alone may be difficult to maintain survival. In this context, methylation of STAT3 by PRMT5 can promote activated tyrosine phosphorylation of STAT3, which may work to maintain continuous STAT3 activation in cancer cells. Furthermore, we also found that PRMT5 plays an important role in the oncogenic pathway induced by another STAT3 activation pathway: SRC. Thus, PRMT5 may maintain STAT3 activation in a variety of cancer types. In addition to these findings, PRMT5 expression itself was induced by STAT3. The existence of such a positive feedback loop maintains bistability by supporting the activated state⁶⁸. Thus, this positive activation loop may be important for accelerating and maintaining STAT3 activity in cancer cells. In addition, methylation of SMAD7 by PRMT5 can reportedly promote the binding of SMAD7 to IL-6 receptor glycoprotein 130, resulting in induction of STAT3 activation. Additionally, PRMT5 inhibition can suppress tumour growth of lung cancer cells in mice⁶⁹. Yet, as mentioned above, JAK activation alone cannot explain the level of STAT3 activation observed in cancer. It is likely that this mechanism may also be at work with direct STAT3 methylation. In addition, PRMT5 expression levels have been reported to be upregulated in various cancers. PRMT5 can promote cancer through epigenetic regulation via histone methylation or through activating cancer-related proteins, such as C-MYC and NF-kB, and inhibiting tumour suppressor p53 by direct methylation^{37, 70, 71}. In this study, we found that STAT3 can induce PRMT5 expression, suggesting that cancer-promoting functions of PRMT5 other than STAT3 activation are involved in the oncogenic function of STAT3. Therefore, further analysis of the regulation of other oncogenic signals and cancer-associated transcription factors via PRMT5 induced by STAT3 will provide insights into the molecular mechanisms underlying the oncogenic functions associated with the PRMT5/STAT3 activation loop.

To date, a variety of STAT3-targeted cancer therapies have been developed²⁴. However, there are no clinically applied drugs that inhibit STAT3 itself. At present, clinical trials of PRMT5 inhibitors are underway⁷², but it is unclear whether they will be safe and effective given the broad physiological effects of PRMT5³⁷. Thus, the activation of STAT3 by the PRMT5/MEP50/STAT3 complex discovered in this study may be a new and specific target for suppressing STAT3 for cancer therapeutic approaches.

Cell culture

HEK293T and HeLa cells were purchased from the JCRB Cell Bank; Lenti-X 293T cells were purchased from Takara Bio (Japan); NIH3T3, BEAS2B, A549, and HCC827 cells were purchased from American Tissue Culture Collection (ATCC). p53 knockout mouse embryonic fibroblasts (p53^−/− MEFs) were obtained as described elsewhere. Mycoplasma contamination was routinely monitored using MycoStrip (InvivoGen, San Diego, CA). HEK293T, HeLa, NIH3T3, and p53^−/− MEFs were cultured in Dulbecco’s Modified Eagle Medium (DMEM: Nacalai Tesque) supplemented with 10% foetal bovine serum (FBS; Nichirei Biosciences, Japan). Lenti-X 293T cells were cultured in DMEM (high glucose) supplemented with 10% FBS. BEAS2B cells were cultured in DMEM/F-12K (Nacalai Tesque, Japan) supplemented with 10% FBS. A549 and HCC827 cells were cultured in RPMI-1640 medium (Nacalai Tesque) supplemented with 10% FBS. Streptomycin (100 U/ml)/Penicillin-G (100 µg/ml) solution (Nacalai Tesque) was added to all cell culture media.

Reagents, antibodies, and plasmid construction

Recombinant IL-6, recombinant soluble IL-6 receptor (sIL-6R), and recombinant HGF were purchased from R&D Systems (Minneapolis, MN). Polybrene was purchased from Nacalai Tesque. Puromycin and hygromycin were purchased from Sigma-Aldrich. Doxycycline was purchased from FUJIFILM Wako Pure Chemical. For stable luciferase expression, CMV-GFP-T2A-luciferase plasmid was purchased from System Biosciences. The antibodies used in this study are listed in Supplementary Table 1. For shRNA construction, synthesized oligo was inserted into the pLKO.1 puro vector (Addgene: plasmid #8453). For inducible shRNA expression, the synthesized oligo was inserted into the Tet-pLKO-puro vector (Addgene: plasmid #21915). For STAT3 knockout using the CRISPR/Cas9 system, the synthesized oligo was inserted into the pLenti-CRISPR-V2-Puro vector (Addgene: #98290). The target sequences for RNAi-mediated knockdown are listed in Supplementary Table 2. The target sequences for CRISPR/Cas9-mediated knockout are listed in Supplementary Table 3. Myc-tagged STAT3 mutants were generated by PCR, and the relevant primers are listed in Supplementary Table 4. Myc-tagged STAT3 and its mutant cDNAs were inserted into the pcDNA3 vector (Thermo Fisher Scientific, Waltham, MA) or pLVSIN-Hyg vector (Takara Bio) using the In-Fusion HD cloning kit (Takara Bio) according to the manufacturer's instructions.

Transfection, recombinant virus production, and infection

The expression plasmid was transfected into cells using GeneJuice (Merck Millipore) according to the manufacturer’s instructions for transient gene expression. Recombinant retrovirus production was performed as described previously³⁴. Recombinant lentivirus was produced in Lenti-X 293T cells by transfection of the expression plasmid and Lentiviral High Titer Packaging Mix (Takara Bio) using TransIT-Lenti transfection reagent (Mirus Bio) according to the manufacturer’s instructions. Twenty-four hours after transfection, the medium was changed and the cells were incubated for an additional 24 hours. The lentivirus-containing medium was collected, then debris was removed using a syringe filter (pore size 0.45 µm; CORNING, Corning, NY). The filtered culture media were supplemented with 8 µg/mL polybrene to infect cells. Infected cells were selected by puromycin (2.5 µg/mL; Sigma-Aldrich, St. Louis, MO) or hygromycin (200 µg/mL; Sigma-Aldrich).

Immunoprecipitation, cell fractionation, and immunoblotting

Cells were lysed in TNE lysis buffer (1% Nonidet P-40, 10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, and protease inhibitor cocktail [Nacalai Tesque]). According to the manufacturer's instructions, cell fractionation was performed using the LysoPure Nuclear and Cytoplasmic Extraction Kit (FUJIFILM Wako Pure Chemical). Protein concentrations were measured by Protein Assay Dye Reagent (Bio-Rad, Hercules, CA). Equal amounts of proteins were used for immunoprecipitation or immunoblotting experiments. Immunoprecipitation was performed as described previously³⁵. Cell lysates were incubated with the indicated antibody for 2 hours at 4°C. Protein-G Sepharose beads (Cytiva, UK) were added to the cell lysate, which were then incubated for 2 hours at 4°C. Next, the protein-G beads were collected and washed with lysis buffer before denaturation. SDS-PAGE which was loaded in 30 µg per well was performed, followed by immunoblotting. The proteins separated on the SDS-PAGE gel were transferred to a Poly Vinylidene Fluoride (PVDF) membrane (MERCK, Germany), followed by blocking with 3.5% bovine serum albumin (BSA: Nacalai Tesque) in PBS with 0.05% Tween-20 (FUJIFILM Wako Pure Chemical). The blots were then incubated with the primary antibody for 40 min at room temperature or 4℃ for overnight, and the horseradish peroxidase (HRP)-conjugated secondary antibody for an hour at room temperature (Cytiva). Primary and secondary antibodies were diluted with Bullet Immuno-Reaction Buffer (Nacalai Tesque) or 3.5% BSA (blocking solution). The antibody information was described in Supplementary Table 1. Chemi-Lumi One Ultra (Nacalai Tesque) and LuminoGraph I (ATTO, Japan) were used to visualize the blots. Quantification of protein expression levels were assessed using ImageJ software.

Cell proliferation assay, tumour sphere formation, and CSC population

First, 5×10⁴ cells were seeded per well in a 12-well plate in triplicate. After 24 hours, viable cells were measured by trypan blue excision using a cell counter (BIO-RAD). For tumour sphere formation, 1,000 cells were seeded per well in a 6-well plate ultra-low attachment (CORNING). The cells were maintained in DMEM/F12K supplemented with 20 ng/mL human recombinant epidermal growth factor (EGF: R&D systems) and 20 ng/mL human recombinant basic fibroblast growth factor (bFGF: FUJIFILM Wako Pure Chemical). Seven days after seeding, the tumour sphere with a diameter > 150 µm was counted. The population of CSCs was assessed by FACSCallibur (BD, FranklinLakes, NJ) flow cytometry with an ALDEFLUOR kit (STEM CELL TECHNOLOGY).

RNA isolation, reverse transcription, and quantitative PCR (qPCR)

Total RNA isolation was performed using NucleoSpin RNA plus (Takara Bio), then RNA concentrations were measured using a NanoDrop (Thermo Fisher Scientific). 500 ng RNA was used for reverse transcription with the PrimeScript RT reagent kit (Takara Bio). cDNA templates were subjected to qPCR analysis using the TaqMan Gene Expression Assay Mastermix and TaqMan probes (Thermo Fisher Scientific) with a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). The probe list for the TaqMan gene expression assays is shown in Supplementary Table 5. Each amplification reaction was performed in triplicate, and the average of three threshold cycles was used to calculate the relative amount of transcript in the sample (StepOne software; Thermo Fisher Scientific). Quantification of mRNA was expressed in arbitrary units as the ratio of the sample quantity to the calibrator or the mean values of control samples. All values were normalized to an endogenous control, the human β-actin gene.

Immunofluorescence staining

First, 5×10⁴ cells were seeded per well in a 24-well-glass bottom plate (IWAKI, Japan). Cells were fixed with 4% paraformaldehyde for 15 minutes at 37°C and permeabilized with 0.5% TritonX-100 in PBS for 7 minutes at room temperature. Blocking was performed using 1% BSA, followed by primary antibody incubation (anti-PRMT5 antibody or anti-STAT3 antibody) for 30 minutes at room temperature. Secondary antibodies (Alexa-488-conjugated anti-mouse antibody [Thermo Fisher Scientific] and Alexa-568-conjugated antibody [Thermo Fisher Scientific]) were incubated for 30 minutes at room temperature in the dark. DNA was stained with Hoechst33342 [Thermo Fisher Scientific]. The immunofluorescent image was obtained by IX-70 fluorescence microscopy (EVIDENT, Japan).

Luciferase assay

Luciferase assays were performed as described previously⁷³. According to the manufacturer’s instructions, Cignal Reporter Assay Kit STAT3 (QIAGEN, Germany) was used as a STAT3 reporter plasmid. The HIF-1 reporter plasmid (pGL4.42 [luc2P/HRE/Hygro] vector) was purchased from Promega (Madison, WI), and the NF-kB reporter plasmid used in this study was described⁷⁴. Forty-eight hours after transfection, reporter activity was assessed by GLOWMAX (Promega) with the Dual-luciferase reporter assay system (Promega).

Chromatin immunoprecipitation

V-SRC was expressed by a recombinant retrovirus in HeLa cells. Chromatin was prepared using the ChIP-IT Express Enzymatic kit (Active Motif, Carlsbad, CA). Chromatin immunoprecipitation was performed using anti-STAT3 or -IgG antibodies (Supplementary Table 1). Eluted DNA was analysed by standard PCR techniques. Oligonucleotides used for PCR are described in Supplementary Table 6.

Mouse xenograft model

The animal experiment committee at the Nippon Medical School approved the animal experiments in this study (Approval Number: 29–045, 2022-044), and animal care was conducted following the guidelines for Animal Experiments of Nippon Medical School and the guidelines of The Law and Notification of the Government of Japan⁷⁵, as well as the ARRIVE guidelines. Mice were maintained 12-hour light/12- hour dark cycle at 20–24°C with 40–70% humidity. They were allowed to have free access to standard laboratory mouse chow, MF (Oriental Yeast Co., ltd., Japan), and free access to drinking water. They were housed at a maximum number of five. All mice were checked for stress each day. Nude mice (BALB/c-AJcl nu/nu) were purchased from Japan CREA, Inc (Japan). Luciferase was stably expressed by recombinant lentivirus in cells before transplantation. Then, 1×10⁶ (Fig. 3k, l) or 2×10⁶ (Fig. 5h, i) cells were subcutaneously transplanted into 7-week-old male nude mice (body weight 23–26 g). Tumour volume was assessed using In Vivo Imaging System (IVIS: PerkinElmer). For induction of shRNA expression in xenografted tumours, tumour volume was allowed to reach approximately 150 mm³, then doxycycline (1 mg/mL) was added to the drinking water.

Bioinformatics analysis

Differential expression analysis of PRMT5 between tumour sites and normal sites was performed using the TCGA-LUAD dataset (https://portal.gdc.cancer.gov/projects/TCGA-LUAD) or the TCGA-LUSC dataset (https://portal.gdc.cancer.gov/projects/TCGA-LUSC). The comparison of overall survival rates between the PRMT5 high and low expression groups and the correlation of PRMT5 copy number and PRMT5 expression in LUAD were also examined using the TCGA-LUAD dataset. Datasets were analysed using the UCSC Xena browser (https://xena.ucsc.edu). PRMT5 expression levels across 13 cancer types were examined using TCGA PanCancer Atlas Studies. The Dataset was analysed on cBioPortal (http://www.cbioportal.org). Differential PRMT5 expression in cancer cell lines was examined by the Depmap portal in Cancer Cell Line Encyclopedia (CCLE: https://sites.broadinstitute.org/ccle) at Broad Institute. The ChIP-seq dataset was obtained from the Encyclopedia of DNA Elements at UCSC (ENCODE: https://genome.ucsc.edu/ENCODE/) and was analysed using the UCSC Genome Browser (https://genome.ucsc.edu). The prediction of PRMT5-mediated methylation of STAT3 arginine residues was assessed using PRmePRed (http://bioinfo.icgeb.res.in/PRmePRed/index.html). A prediction score > 0.8 was considered to be a candidate for PRMT5-mediated methylation of arginine residues in STAT3.

Statistical analysis and reproducibility

Microsoft Excel was used for statistical analysis. Results of the in vivo analyses are shown as the mean ± standard error of the mean (SEM), while results of the in vitro analyses are shown as the mean ± standard deviation (SD). Statistical testing was performed using the unpaired two-tailed Student’s t-test. A P-value < 0.05 was considered statistically significant. N numbers and data reproducibility were described in figure legends.

Data availability

All materials used in this study are available with permission from the corresponding author.

Acknowledgements

We thank Wataru Nakajima and Ikuno Uehara for the helpful discussions and Akihiro Yamazaki, Yumi Asano, and Mitsuko Kajita for their technical assistance. We also thank J. Iacona, Ph.D., from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript. This work was supported by JSPS KAKENHI (Grant Numbers 25870792 and 20K09044) and The Kurata Grants by The Hitachi Global Foundation.

Author contributions

T.S., Y.A., and N.T. conceived and designed the experiments. T.S. and Y.A. conducted the experiments with assistance from N.O. Finally, Y.O., N.T., and Y.A. wrote the manuscript. All authors contributed to manuscript reviews.

Competing interests

The authors declare no competing interests.

Supplementary information

Supplementary Information

Description of Additional Supplementary Files

File name: Supplementary Data 1
Description: The source data behind the graph in the main figures.

File name: Supplementary Data 2
Description: The original immunoblot image in the main figures.

File name: Supplementary Data 3
Description: The source data behind the graph in the supplementary figures.

File name: Supplementary Data 4
Description: The original immunoblot image in the supplementary figures.

Conflict of interest: The authors declare that they have no competing interests.

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published 17 May, 2024

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PRMT5-mediated methylation of STAT3 is required for lung cancer stem cell maintenance and tumour growth

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Introduction

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Discussion

Methods

Cell culture

Reagents, antibodies, and plasmid construction

Transfection, recombinant virus production, and infection

Immunoprecipitation, cell fractionation, and immunoblotting

Cell proliferation assay, tumour sphere formation, and CSC population

RNA isolation, reverse transcription, and quantitative PCR (qPCR)

Immunofluorescence staining

Luciferase assay

Chromatin immunoprecipitation

Mouse xenograft model

Bioinformatics analysis

Statistical analysis and reproducibility

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