PKC-ζ mediated reduction of the extracellular vesicles-associated TGF-β1 overcomes radiotherapy resistance in breast cancer
Breast Cancer Research volume 25, Article number: 38 (2023)
Radiotherapy is widely applied in breast cancer treatment, while radiotherapy resistance is inevitable. TGF-β1 has been considered to be an endogenous factor for the development of radiotherapy resistance. As a large portion of TGF-β1 is secreted in an extracellular vesicles-associated form (TGF-β1EV), particularly in radiated tumors. Thus, the understanding of the regulation mechanisms and the immunosuppressive functions of TGF-β1EV will pave a way for overcoming the radiotherapy resistance in cancer treatment.
The superoxide-Zinc-PKC-ζ-TGF-β1EV pathway in breast cancer cells was identified through sequence alignments of different PKC isoforms, speculation and experimental confirmation. A series of functional and molecular studies were performed by quantitative real-time PCR, western blot and flow cytometry analysis. Mice survival and tumor growth were recorded. Student’s t test or two-way ANOVA with correction was used for comparisons of groups.
The radiotherapy resulted in an increased expression of the intratumoral TGF-β1 and an enhanced infiltration of the Tregs in the breast cancer tissues. The intratumoral TGF-β1 was found mainly in the extracellular vesicles associated form both in the murine breast cancer model and in the human lung cancer tissues. Furthermore, radiation induced more TGF-β1EV secretion and higher percentage of Tregs by promoting the expression and phosphorylation of protein kinase C zeta (PKC-ζ). Importantly, we found that naringenin rather than 1D11 significantly improved radiotherapy efficacy with less side effects. Distinct from TGF-β1 neutralizing antibody 1D11, the mechanism of naringenin was to downregulate the radiation-activated superoxide-Zinc-PKC-ζ-TGF-β1EV pathway.
The superoxide-zinc-PKC-ζ-TGF-β1EV release pathway was elucidated to induce the accumulation of Tregs, resulting in radiotherapy resistance in the TME. Therefore, targeting PKC-ζ to counteract TGF-β1EV function could represent a novel strategy to overcome radiotherapy resistance in the treatment of breast cancer or other cancers.
Trial registration: The using of patient tissues with malignant Non-Small Cell Lung Cancer (NSCLC) was approved by the ethics committees at Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (NCC2022C-702, from June 8th, 2022).
Breast cancer is the leading diagnosed cancer in women and ranks the top incidence among all cancers. It is reported that up to 83% of breast cancer patients have received radiotherapy (RT) . However, data over the last decade have shown that RT inevitably induces the immunosuppressive tumor microenvironment (TME), which in turn aggravates the incidences of radiotherapy resistance .
In the TME, transforming growth factor beta 1 (TGF-β1) is an immunosuppressive cytokine that plays an important role in the differentiation and development of Treg population [3,4,5]. Although the clinical data have shown that the local TGF-β1 levels in tumors would significantly increase after radiotherapy , it is surprising that no fluctuation of peripheral TGF-β1 has been observed [7, 8]. These findings indicate a distinct form of TGF-β1 in the breast cancer tissues that contribute to the local accumulation of TGF-β1. Traditionally, TGF-β1 is known to be secreted as latency-associated peptide-TGF-β1 (L-TGF-β1), which needs to be released from its binding proteins to form free TGF-β1 before it binds TGF-β1 receptor (TGF-βR) to activate downstream signaling pathways . Recent studies, however, have revealed that, with the help of integrin ανβ8, TGF-β1 can expose the active site and binds directly to TGF-βR in its precursor form . Moreover, a third form of TGF-β1 has been found as the extracellular vesicle (EV) associated-TGF-β1 (here after referred as TGF-β1EV) [11, 12]. In contrast to free TGF-β1 or L-TGF-β1, the functional TGF-β1EV can transmit signals rapidly and effectively through endocytosis . We then suppose that the elevated intratumoral TGF-β1 post-radiotherapy are associated with EVs, which cannot effectively diffuse into blood circulation due to their relatively large sizes .
Despite RT has been thought to promote anti-tumor immunity [15, 16], it is also reported to induce of Treg differentiation by increasing the intratumoral TGF-β1 level within the TME [17, 18]. As a major immunosuppressive regulator, the recruitment and accumulation of Tregs within TME undermines spontaneous T cell activation , resulting in higher risks of tumor aggressiveness, recurrence, and metastasis , as well as the induction of resistance to radiotherapy, and finally patients’ shorter survival [9,10,11,12,13]. Thus, TGF-β1 has been considered to be an endogenous factor for the development of RT resistance . However, it is still unknown whether RT induced-TGF-β1 is in EV-associated form. Therefore, understanding the existing form of TGF-β1 and the immunosuppressive mechanisms of TGF-β1EV in the radiated tumors could shed light on overcoming radiotherapy resistance.
Our previous work has shown that a natural flavonoid, naringenin is capable to reduce TGF-β1 secretion from breast cancer cells through inhibiting phosphorylation levels of PKCs . In the present study, it is of strong necessity to explore whether naringenin exerts the same effect on reducing the secretion of TGF-β1EV in the radiated tumors. As distinct PKCs have been reported to play different roles in the process of vesicles secretion in various kinds of cells [23,24,25,26], specific types of PKCs may regulate the secretion of TGF-β1EV in breast cancer cells. We used a murine triple negative breast cancer model to demonstrate that irradiation promoted the release of TGF-β1 from the cancer cells, and a large proportion of the secreted TGF-β1 was in the extracellular vesicle-associated form.
Excitedly, we found that the expression of PKC-ζ was preferentially enhanced by irradiation and the blockage of PKC-ζ restricted the TGF-β1EV secretion, indicating that PKC-ζ contributed to the releasing of TGF-β1EV. More importantly, naringenin, but not 1D11, significantly improved the radiotherapy efficacy with low side effects. The underlying mechanism of naringenin was via downregulating of the superoxide-Zinc-PKC-ζ-TGF-β1EV pathway activated by radiation. Therefore, targeting PKC-ζ to counteract TGF-β1EV function could represent a novel strategy to overcome radiotherapy resistance in the treatments of breast cancers or other cancers.
Materials and methods
Experimental animal and cell lines
Female BALB/c (6–8 weeks old) were purchased from Vital River Laboratory Animal Technology (Beijing, China). Foxp3-GFP mice were kindly provided by Prof. Yangxin Fu (University of Texas, Southwestern Medical Center, Texas, USA). All animal experiments were performed according to the institutional ethical guidelines on animal care and the protocols used for this study were approved by the Animal Care and Use Committee at the Institute of Biophysics, Chinese Academy of Sciences. Murine breast cancer 4T1 cell line was obtained from ATCC and cultured in 5% CO2 and maintained in RPMI 1640 medium supplemented with 10% FBS (VivaCell, Isreal) 100 U/ml penicillin, and 100 mg/ml streptomycin.
Exocellular vesicles purification
For in vitro experiment, 4T1 cells were cultured in RPMI 1640 media supplemented with 10% EV-depleted FBS. Supernatant was collected and centrifuged at 500 g for 10 min followed by a step of 3000 g for 20 min at 4 °C to pellet cells and debris. The supernatant was collected without disturbing the cell/debris pellet and was transfered to an ultracentrifuge tube. Then the supernatant was centrifuged at 100,000 g for 70 min at 10 °C and the EV pellets were collected. The pellets were resuspended in a small volume of PBS. For tumor tissues, the harvested tumors were dissected and cut into small pieces, followed by culture in RPMI 1640 media supplemented with 10% EV-depleted FBS for 48 h. Supernatant of tumor pieces was collected for the EVs purification. The EVs was characterized by Transmission Electron Macroscopy (FEI, Tecnai Spirit, 120 kV, USA) and quantified by BCA protein assay kit.
TGF-β1 detection by ELISA
TGF-β1 in supernatant or EV was detected by ELISA (DY1679, R&D Systems, Minneapolis, MN). In brief, the 96-well microplate was coated with the Capture antibody overnight at 4 degree. Cells were washed by filling each well with Washing buffer. The plate was blocked by Block buffer for 1 h. TGF-β1 was activated by HCl and added to each well and incubate 2 h at room temperature. Cells were washed and the detection antibodies were added and incubated for 2 h. Streptavidin-HRP was added to each well. Washing wells and adding Substrate solution to each well were followed by adding Stop solution. The optical density of each well was determined immediately at 450 nm.
Data sources and processing
The gene mRNA expression matrix and clinical follow-up information of Breast Invasive Carcinoma patients with information of radiation therapy were obtained from the cBioPortal database (http://www.cbioportal.org). The association between TGF-β1 expression and the overall survival was determined using the Kaplan–Meier survival analysis with the 'survival' package (version 4.1.2) in R statistics software, and the Log-rank test was used to detect significant differences. Immune cell infiltration was obtained from the ImmuCellAI database (http://bioinfo.life.hust.edu.cn/ImmuCellAI#!/) and the clinical information was also acquired from the cBioPortal database by using the patient ID.
Cells were lysed by RIPA lysis buffer and the protein concentration was determined by BCA protein assay. Cell lysates were separated by SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were then blotted with the indicated antibodies (anti-TGF-β1 antibody, ab179695; anti-PKCζ antibody, ab108970; anti-p-PKCζ antibody, ab76129; anti-β-actin antibody, ab8226. Abcam, Cambridge, UK).
PMA and CAL treatment
Phorbol myristate acetate (PMA) and Calphostin C (CAL) were purchased from Merck and used at the indicated concentrations. 4T1 cells were treated by PMA or CAL for 48 h. Supernatant was collected and EV was purified.
TGF-β1 knockout of 4T1 cell line generation
gRNA targeting sequence were designed as the sequences described above using CRISPR design tool (https://zlab.bio/guide-design-resources) and the sgRNA oligos with BbsI restriction site were prepared by annealing. The gRNA oligos were constructed into pX458M and EZ-Guide plasmids, respectively. Digest pX458M-gRNA1 and EZ-Guide-gRNA2 plasmid using XhoI and HindIII Restriction Enzyme (New England Biolabs, Beijing, China) and the two plasmids were ligated by T4 DNA ligase. pX458M-gRNA1 + gRNA2 plasmid were transformed into 4T1 cells by Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA). After 48 h transfection, GFP + cells were sorting into 96-well plate by FACS Influx (BD, Franklin Lake, NJ) and cells were identified by qPCR for TGFB mRNA and ELISA for TGF-β1 protein.
Quantitative real-time PCR (qPCR) assay
Total RNA was isolated from cells by Trizol (Invitrogen, Carlsbad, CA). The mRNA was reversely transcribed to cDNA by M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA). qPCR was performed using SYBR Green qPCR SuperMix (Invitrogen, Carlsbad, CA) to detect the expression of TGF-β1 and PKCs mRNA. Gene expression was normalized to GAPDH expression and presented as fold-change compared to the Control experiment.
Coculture of EVs and naïve splenocytes
Anti-CD3 antibody was coated into 96-well U plate overnight. Naïve splenocytes were added into each well. EVs were isolated from cells treated by different reagents and quantified by BCA protein assay. Different EVs were diluted by same times and added to naïve splenocytes with anti-CD28 antibody. After 72 h, cells were collected, stained with fluorescent antibodies and detected by Flow cytometry.
Immunohistochemical studies were done in 5-µm sections of paraffin-embedded tumor tissues using antibodies for TGF-β1 to determine its content in the tumors. The slides were incubated in citrate buffer for 20 min in a steamer and endogenous peroxidase was blocked by incubation with 3% H2O2 for 20 min at room temperature. The anti-TGFβ1 antibody (ab179695, Abcam, Cambridge, UK) was used in a dilution of 1:500. The slides were then stained with the secondary antibody in a dilution of 1:500. To determine the protein expression, stained slides were examined under fluorescence microscopy.
4T1 cells at 80% confluence were transfected with siRNA (sc-36254, Santa Cruz) by Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA) for 24 h. Cells were treated by PMA or radiation for indicated time.
Treg differentiation assay
Spleen of GFP-Foxp3 transgenic BALB/c mice was harvested and single cell suspension was prepared. Cells were treated with anti-CD3ε/CD28 function antibody and EV or TGF-beta1 or 1D11 (BE0057, InVivoMab, West Lebanon, NH) for 72 h. Percentage of CD4+GFP+ cells in CD4+ cells was detected by BD FACSCalibur.
The following putative structures of mouse PKCs were downloaded from the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/ and used in our study: KPCA (UniProtKB: P20444), KPCB (UniProtKB: P68404), KPCD (UniProtKB: P28867), KPCE (UniProtKB: P16054), KPCG (UniProtKB: P63318), KPCI (UniProtKB: Q62074), KPCL (UniProtKB: P23298), KPCT (UniProtKB: Q02111), KPCZ (UniProtKB: Q02956). Geometrical alignments, as well as visualization, were performed with PyMOL version 2.1.0.
Zinc-specific fluorescence staining
Before radiation administration, 4T1 cells were treated with Naringenin of 200 uM concentration for 30 min. X-Ray of 2, 4 or 8 Gy dose was used to treat 4T1 cells, respectively. After 2 h, cells were collected and washed with PBS 3 times. TSQ was dissolved in Lock’s buffer (pH 7.4) and cells were stained with 150 nM TSQ for 1 min. After 3 times washing, cells were added into 96-well plate and were examined under a fluorescence microplate reader (Excitation, 345 nm; Emission 495 nm) (SpectraMax M4 Multi-Mode Microplate Reader, Molecular Devices, San Jose, CA). The instrument measures the intensity of the reradiated light and expresses the result in Relative Fluorescence Units (RFU) using SoftMax Pro Software (Standard Edition 7.1).
4T1 cells were subcutaneously injected at 5 × 104 cells per mouse. Mice were randomized to treatment groups. Radiotherapy was administrated with a dose of 10 Gy when tumor volume reached 70–80 mm3. 100 mg/kg naringenin was administrated daily by intragastric administration for 30 days. 5 mg/kg 1D11 was injected intraperitoneally 3 times per week for 3 weeks. Tumor volumes were measured twice a week and calculated as length*width*height/2. The survival days of tumor-bearing mice were recorded. All animal experiments were performed according to the institutional ethical guide lines on animal care and the protocols used for this study were approved by the Animal Care and Use Committee at the Institute of Biophysics, Chinese Academy of Sciences.
Flow cytometry and antibodies
Single-cell suspensions were prepared. Samples were stained (20–30 min) with the following antibodies: anti-CD45, anti-CD3, anti-CD4, anti-CD8α, anti-CD25 antibodies. For intracellular staining, cells were fixed, permeabilized overnight at 4 °C (Fixation/Permeabilization Concentrate and Diluent kit, eBioscience, San Diego. CA) and subsequently stained using anti-Foxp3 antibody for 30 min. All experiments were performed on BD FACSCalibur or BD LSRFortessa and data was analyzed with FlowJo 7.6.1.
Student’s t test was used for comparisons of datasets with two groups. For multiple comparisons, we used type II ANOVA with correction of statistical hypothesis testing. Statistical significance was considered reached for p values < 0.05. Survival was analyzed by Log-rank (Mantel-Cox) test.
TGF-β1 neutralizing antibody 1D11 only partially improved the radiotherapy effect on 4T1 breast cancer
As RT has been reported to cause the increases of the intratumoral TGF-β1 level and the infiltration of Tregs in the TME [17, 18], TGF-β1 therefore is considered to be an endogenous factor for RT resistance. To investigate how much TGF-β1 is secreted in the form associating with EVs in clinical settings, the EVs of tumor samples were isolated from six NSCLC patients, respectively. As expected, up to 72% of TGF-β1 was secreted in the EV-associated form in these tumors from the NSCLC patients (Fig. 1A).
Given the clinical data indicate that the TGF-β1 level is increased in the irradiated tumors [8, 27], we then wondered if the increased TGF-β1 could augment Tregs infiltration in tumors after irradiation. In line with the previous finding, the irradiation was found to promote more Tregs infiltration in 4T1 tumors than the untreated control (Fig. 1B, C). Curiously, compared to RT alone, the combination of TGF-β1 neutralizing antibody 1D11 only partially suppressed radiation-induced Tregs infiltration (Fig. 1C). A delayed tumor growth was observed after RT and 1D11 combination treatment (Fig. 1D), while the survival benefit was comparable to the mice received RT alone (Fig. 1E, F). These results suggested that 1D11 did not effectively block the function of the TGF-β1EV to improve the effect of radiotherapy.
TGF-β1 in tumor-derived EVs induce Treg differentiation
In order to verify the functions of the TGF-β1EV secreted by breast cancer cells, we isolated EVs from 4T1 cells and characterized. The results showed that the average diameter of the EVs was about 40 nm (Fig. 2A), and approximately 82% of TGF-β1 secreted by 4T1 cells was associated with the EVs (Fig. 2B). Consistent with the finding in 4T1 cells, up to 80% of the TGF-β1 was secreted with EVs from 4T1 tumor tissues of balb/c mice (Fig. 2C). Notably, we found that the TGF-β1EV was mainly in the form of latent-TGF-β1, while the TGF-β1 left in the EV-free supernatant derived from 4T1 cells was mainly in the form of active-TGF-β1 (Fig. 2D). The latent TGF-β1EV transmits signals after endocytosis [13, 28], which probably explained why 1D11 or other TGF-β1 inhibitors to bind ligands or receptors could not effectively block the function of the TGF-β1EV.
To further determine if the TGF-β1EV affect T cells differentiation, we generated the TGF-β1 knockout (KO) 4T1 cell line (Fig. 2E, F). A significant increase of Tregs population in response to the EVs from wild type (WT) 4T1 cells were observed. The TGF-β1 knockout impeded the ability of the EVs to induce Tregs population (Fig. 2G). However, 1D11 only had a partial effect on the TGF-β1EV-induced Tregs infiltration (Fig. 2I). We further demonstrated that the EVs from 4T1 cells directly promoted the differentiation of splenic CD4+ T cells into Tregs (Additional file 1: Fig. S1A). In addition, the TGF-β1EV did not show any effects on the proliferation and apoptosis of Treg cells (Additional file 1: Fig. S1B, C). Interestingly, the PHK67-labeled TGF-β1EV was rapidly engulfed by CD4+CD25+ lymphocytes and resulted in the intracellular accumulation (Fig. 2H). These data suggested that the tumor-derived EVs containing TGF-β1 is capable of inducing CD4+ T cells differentiation into Tregs, which cannot be effectively blocked by the TGF-β1 neutralizing antibody.
Increased TGF-β1EV correlates with Tregs accumulation in the TME
To test if the TGF-β1EV contributes to Tregs induced radiotherapy resistance in the TME, we employed a 4T1 breast cancer model treated by irradiation (Fig. 3A). In line with the previous reports , the area of TGF-β1 positive region in IHC slides was increased from 6.4% (on day 16) to 26.8% (on day 28) after tumor cell injection (Fig. 3B, C). Notably, irradiation further enlarged the area of TGF-β1 positive region from 25% (on day 2) to 45% (on day 14) after RT (Fig. 3B, C). However, the TGF-β1 concentration in peripheral blood was not changed regardless of whether the mice were irradiated (Fig. 3D, E). The irradiation-induced TGF-β1 in tumor was supposed to be also mainly in a form associated with EVs. Indeed, more than 65% of the TGF-β1 was on the EVs in both 4T1 cells and tumor tissues after irradiation (Additional file 1: Fig. S2A, B). Furthermore, compared with the control group, the irradiation promoted 4T1 tumors to release more TGF-β1EV (Fig. 3F), thus resulting in more Tregs infiltration (Fig. 3G). To verify if the TGF-β1EV tends to retain in TME, we intratumorally injected the fluorescent-labeled TGF-β1EV and the free TGF-β1 into 4T1 tumor, respectively. The results demonstrated that the free TGF-β1 diffused away from tumor tissue much faster than the TGF-β1EV (Fig. 3H). Compared with 1 h post-injection, only 53% of free TGF-β1 remained after 24 h of injection, meanwhile, much higher fluorescent intensity (~ 74%) was retained in tumors after the TGF-β1EV injection (Fig. 3I). These results suggested that the retained TGF-β1EV in TME highly possibly induced immunosuppression through promoting the differentiation of CD4+ T cells to Tregs.
PKC-ζ masters TGF-β1EV secretion upon radiation in 4T1 cells
As PKCs have been reported to be involved in the secretion of EVs, to understand the underlying mechanism of the TGF-β1EV secretion, we focused on PKCs. As expected, the pan-PKCs agonist PMA promoted the TGF-β1EV secretion from 4T1 cells. Conversely, the inhibition of PKCs by Calphostin C (CAL) showed an opposite effect (Fig. 4 A, B).
Radiation has been shown to promote the TGF-β1EV secretion from tumor tissues (Fig. 3F). To identify the subtype of PKCs regulating the TGF-β1EV secretion, we compared the mRNA expression of various PKC subtypes before and after irradiation. We found that, even after receiving a low dose of radiation as 2 Gy, the mRNA expression of PKC-ζ was elevated more significantly than that of other PKC subtypes (Fig. 4C), suggesting that PKC-ζ could effectively regulates the TGF-β1EV secretion. We then used the siRNA of PKC-ζ, which was verified for its effectiveness (Additional file 1: Fig. S3), to treat 4T1 cells before treatment of PMA or radiation. The results showed that the suppression of PKC-ζ by siRNA significantly inhibited the TGF-β1EV secretion induced by PMA (Fig. 4D, E). As expected, the secretions of EVs and TGF-β1EV from 4T1 cells (Fig. 4F, G) and the phosphorylation level of PKC-ζ in 4T1 cells (Fig. 4J) were dose-dependently enhanced after irradiation. Accordingly, the PKC-ζ siRNA significantly reduced the TGF-β1EV release induced by irradiation (Fig. 4H, I). These results indicated that PKC-ζ could play an essential role in the TGF-β1EV secretion.
Naringenin reduces TGF-β1EV secretion by inhibition of PKC-ζ on phosphorylation level
We have previously demonstrated that a natural flavonoid, naringenin, can decrease total TGF-β1 secretion through regulation of PKCs . Deservedly, naringenin might reduce the secretion of TGF-β1EV via inhibiting PKC-ζ phosphorylation. Here, our results showed that naringenin had a dose-dependent effect on reducing the level of phosphorylation of PKC-ζ stimulated by irradiation (Fig. 5A) but had no effects on the level of PKC-ζ mRNA expression (Fig. 5B) in 4T1 cells. Furthermore, naringenin markedly attenuated the secretions of the EVs and the TGF-β1EV promoted by radiation (Fig. 5C, D), thus in turn resulting in the prevention of the TGF-β1EV-induced Tregs differentiation from CD4+ T cells (Fig. 5E). In addition, the in vivo data further demonstrated that naringenin reduced the TGF-β1EV secretion from the irradiated 4T1 tumors (Fig. 5F), indicating that naringenin could be used as an inhibitor of PKC-ζ phosphorylation to combine with radiotherapy as a promising strategy for breast cancer treatment.
Naringenin reduces PKC-ζ phosphorylation via inhibiting its superoxide-induced release of zinc
To further investigate the regulatory mechanism of naringenin on the TGF-β1EV release when it combined with RT, we need to answer what caused the elevation of PKC-ζ phosphorylation after radiation. It has been reported that PKC can be activated by the exposure to oxidants . In rat brain, the phosphorylated activation of PKC has been thought via oxidation of thiols and release of zinc from the cysteine-rich region . We then assumed that radiation could promote superoxide production, which stimulated PKC-ζ activity via the release of zinc from zinc finger domain of PKC-ζ in breast cancer cells. When the sequences of PKCs were aligned, it showed that, unlike other PKCs, the atypical PKCs (ζ and ι) have only one single zinc finger motif of the cysteine-rich region in the conserved C1 domain (Fig. 6A). The atypical PKCs also has the smallest number of cysteines in zinc finger region among all PKC isoforms (Fig. 6B). However, the two zinc finger motifs in either classical or novel PKCs are tightly bound to each other leading the cysteine residues are partially hidden within the cleft of the binding interface, but the individual cysteine-rich region for the atypical PKCs is highly isolated from other residues (Additional file 1: Fig. S4A, B). Considering that the superoxide has a very short half-life to stimulate the autonomous PKC activity , we assume that the atypical PKCs including the ζ and ι isoforms are susceptible for superoxide-induced thiol oxidation and following activity increase. The mRNA relative expression of PRKCZ was significantly higher than PRKCI in breast cancer cells, including 4T1 cells (Fig. 6C) and MDA-MB-231 cells (Additional file 1: Fig. S5A), which indicating PKC-ζ may be activated by superoxide more easily than PKC-ι.
Radiation is reported to promote TGF-β1 activation through reactive oxygen species (ROS), which is mostly composed of superoxide radicals (O2˙−) . Here we demonstrated that irradiation induced a time-dependent increasing level of O2˙- (DHE staining) (Fig. 6D), a dose-dependent increase of zinc release (Fig. 6F), and an elevated level of PKC-ζ phosphorylation (Fig. 4J), suggesting a positive correlation among O2˙−, zinc release and PKC-ζ phosphorylation. When 4T1 cells were treated with paraquat (PQ), one of the superoxide generators, a marked elevation of O2˙− was observed. Similar to superoxide dismutase (SOD), naringenin reduced the level of O2˙− induced by irradiation or PQ to a relatively low level in a dose dependent manner (Fig. 6D, E). By reducing of the elevated level of O2˙−, naringenin inhibited zinc release (Fig. 6F, Additional file 1: Fig. S5B) and reduced PKC-ζ phosphorylation level significantly (Fig. 6G, H), demonstrating that naringenin decreases the TGF-β1EV release via inhibition of the superoxide-Zinc-PKC-ζ-TGF-β1EV pathway (Fig. 6I). As one of the top transcription factors for PRKCZ gene expression (PROMO analysis), NF-kB showed a radiation induced nucleus translocation, which was modulated by naringenin (Additional file 1: Fig. S6). The results suggested that RT induced PKC-ζ activation and then feedback stimulated PRKCZ mRNA expression via NF-kB nuclear binding.
PKC-ζ inhibition overcomes the TGF-β1EV mediated radiotherapy resistance
To evaluate the in vivo therapeutic effects of naringenin combined with RT, the 4T1 tumor model was used (Fig. 7A). Compared with RT alone, RT combined with naringenin significantly decreased radiation-induced Tregs infiltration, resulting in a higher ratio of CD8+/Treg (Fig. 7B, C). The reduction of suppressive Treg cells in the TME by naringenin could bring an inhibition of tumor weights (Fig. 7D) and a prolonged survival of mice bearing breast tumors (Fig. 7E, F). When compared to 1D11 antibody, naringenin combined with RT was more effective in delaying the tumor growth (Fig. 7G). After RT, the body weights of mice decreased dramatically. The combination of naringenin could restore somewhat of mice weight on the 19 day, 24 day and 37 day after tumor inoculation (Fig. 7H). Specifically, the weights of three mice among ten were lower than 15 g after the mice received RT combining with 1D11 treatments. In contrast, naringenin quickly recovered the mice weight loss caused by radiation and no mouse weight was lower than 15 g (Fig. 7I). A much shorter time was needed to recover for the mice treated by RT combined with naringenin than that with 1D11 (Fig. 7J). Therefore, the inhibition of PKC-ζ to prevent the TGF-β1EV secretion could be an optional combination strategy to overcome the radiotherapy resistance.
Although RT is widely applied to breast cancer patients, radiotherapy resistance is inevitable, presented as the tumor recurrence and poor prognosis. Here we demonstrated that the irradiation preferred to induce an elevated expression and phosphorylation level of PKC-ζ within the TME, resulting in the resistance to radiotherapy by promoting the TGF-β1EV secretion. An effective way to intervene the TGF-β1EV release from breast cancer cells was first put forward in this study.
In clinic, an elevated TGF-β1 level induced by irradiation has been frequently found accumulation in tumor tissues but not in the circulating system , suggesting the TGF-β1 is mainly associated with EVs. Not only in breast cancers of mice, but also in human NSCLC tissues, large proportion of TGF-β1 was found associated with the EVs. The fact indicates that TGF-β1 in other types of human cancers may exist in a similar form, which makes the TGF-β1EV widely adapted for other cancers as a promising biomarker. It shows that the high level of intra-tumoral TGF-β1 is usually accompanied with the poor prognosis. Therefore, the TGF-β1EV can also be used as a tumor tissue biomarker to personalize radiation therapy for breast cancers.
Different inhibitors targeting the TGF-β1 pathway have been developed to synergize with radiotherapy in clinical trials, however, the combinational treatments are sometimes with limited efficacies and even less effective outcomes . Here we demonstrated that RT treatment effectively controlled tumor growth but promoted more TGF-β1EV release. Different to the free form TGF-β1, the TGF-β1EV can be endocytosed by receipt cells and transmit intracellular signaling effectively. The failure of the TGF-β1 antibody for the TGF-β1EV blockage possibly resulted in the low efficiency to overcome the TGF-β1EV mediated immunosuppression. Therefore, selective inhibition of the TGF-β1EV release is a promising combination therapeutic strategy for preventing breast cancer progression.
PKC family members are reported to be activated early upon irradiation . Our data provide the further evidence on the importance of superoxide activating PKC-ζ for the TGF-β1EV release, and how naringenin intervene in this process. Although further studies are still needed to determine how naringenin regulates oxidation, our data suggest that naringenin may transform the O2˙- induced by radiation to a non-toxic form, such as H2O2 , and maintain the killing ability of oxidation to tumors. In the study, we demonstrated that irradiation induced a significant increase of PKC-ζ expressions on three levels, including the level of transcription, translation and phosphorylation. Naringenin, however, could block radiotherapy-induced PKC-ζ only on the phosphorylation level to enhance tumor control (Figs. 5A–D and 7). The data indicated that the phosphorylation level of PKC-ζ was particularly important in controlling the release of TGF-β1EV.
Preciously, we have demonstrated that naringenin inhibits TGF-β1/Smad3 signaling pathway via the decrease of smad3 expression and can directly block receptor interaction [35, 36], leading to the reduction of Tregs production. Naringenin (YPS345) has been proved to effectively relieve radiation induced pulmonary inflammation and fibrosis , which is currently in an ongoing phase II clinical trial in China (NO. CTR20212450). Based on the evidence that naringenin could relieve RT induced toxicity and improve the effectiveness, naringenin is expected to elevate the radiation dose necessary for killing tumors, meanwhile minimizing the side effects of irradiation and overcoming the radiation therapy resistance via the TGF-β1EV intervention. In summary, our data substantiate naringenin to be a promising candidate for the development of potential anti-TGF-β1 agents to overcome radiotherapy resistance.
Availability of data and materials
All data associated with this study and/or analyzed during the current study are available from the corresponding author.
Transforming growth factor-β1
Protein kinase C
Protein kinase C zeta
Regulatory T cells
- TGF-β1EV :
Extracellular vesicle associated-TGF-β1
The cancer genome atlas
Non-small cell lung cancer
Enzyme linked immunosorbent assay
Phorbol myristate acetate
Reactive oxygen species
Delaney G, Jacob S, Featherstone C, Barton M. The role of radiotherapy in cancer treatment: estimating optimal utilization from a review of evidence-based clinical guidelines. Cancer. 2005;104:1129–37.
Barker HE, Paget JT, Khan AA, Harrington KJ. The tumour microenvironment after radiotherapy: mechanisms of resistance and recurrence. Nat Rev Cancer. 2015;15:409–25.
Chen W, Jin W, Hardegen N, et al. Conversion of peripheral CD4+CD25- naive T cells to CD4+CD25+ regulatory T cells by TGF-beta induction of transcription factor Foxp3. J Exp Med. 2003;198:1875–86.
Liu Y, Zhang P, Li J, Kulkarni AB, Perruche S, Chen W. A critical function for TGF-beta signaling in the development of natural CD4+CD25+Foxp3+ regulatory T cells. Nat Immunol. 2008;9:632–40.
Farhood B, Khodamoradi E, Hoseini-Ghahfarokhi M, et al. TGF-β in radiotherapy: mechanisms of tumor resistance and normal tissues injury. Pharmacol Res. 2020;155:104745.
Barthelemy-Brichant N, Bosquée L, Cataldo D, et al. Increased IL-6 and TGF-beta1 concentrations in bronchoalveolar lavage fluid associated with thoracic radiotherapy. Int J Radiat Oncol Biol Phys. 2004;58:758–67.
Aula H, Skyttä T, Tuohinen S, et al. Decreases in TGF-β1 and PDGF levels are associated with echocardiographic changes during adjuvant radiotherapy for breast cancer. Radiat Oncol. 2018;13:201.
Stanojković TP, Matić IZ, Petrović N, et al. Evaluation of cytokine expression and circulating immune cell subsets as potential parameters of acute radiation toxicity in prostate cancer patients. Sci Rep. 2020;10:19002.
Khalil N. TGF-beta: from latent to active. Microbes Infect. 1999;1:1255–63.
Campbell MG, Cormier A, Ito S, Seed RI, Bondesson AJ, Lou J, et al. Cryo-EM reveals integrin-mediated TGF-β activation without release from latent TGF-β. Cell. 2020;180:490–501.
Clayton A, Mitchell JP, Court J, Mason MD, Tabi Z. Human tumor-derived exosomes selectively impair lymphocyte responses to interleukin-2. Cancer Res. 2007;67:7458–66.
Shelke GV, Yin Y, Jang SC, Lässer C, Wennmalm S, Hoffmann HJ, et al. Endosomal signalling via exosome surface TGFβ-1. J Extracell Vesicles. 2019;8:1650458.
Ringuette Goulet C, Bernard G, Tremblay S, Chabaud S, Bolduc S, Pouliot F. Exosomes induce fibroblast differentiation into cancer-associated fibroblasts through TGFβ signaling. Mol Cancer Res. 2018;16:1196–204.
Xiong R, Vandenbroucke RE, Broos K, et al. Sizing nanomaterials in bio-fluids by cFRAP enables protein aggregation measurements and diagnosis of bio-barrier permeability. Nat Commun. 2016;7:12982.
Weichselbaum RR, Liang H, Deng L, Fu YX. Radiotherapy and immunotherapy: A beneficial liaison? Nat Rev Clin Oncol. 2017;14(6):365–79.
McLaughlin M, Patin EC, Pedersen M, Wilkins A, Dillon MT, Melcher AA, Harrington KJ. Inflammatory microenvironment remodelling by tumour cells after radiotherapy. Nat Rev Cancer. 2020;20(4):203–17.
Arina A, Beckett M, Fernandez C, et al. Tumor-reprogrammed resident T cells resist radiation to control tumors. Nat Commun. 2019;10:3959.
Cytlak UM, Dyer DP, Honeychurch J, Williams KJ, Travis MA, Illidge TM. Immunomodulation by radiotherapy in tumour control and normal tissue toxicity. Nat Rev Immunol. 2022;22:124–38.
Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self. Nat Immunol. 2005;6:345–52.
Su S, Liao J, Liu J, Huang D, He C, Chen F, et al. Blocking the recruitment of naive CD4(+) T cells reverses immunosuppression in breast cancer. Cell Res. 2017;27:461–82.
Kim SJ, Letterio J. Transforming growth factor-beta signaling in normal and malignant hematopoiesis. Leukemia. 2003;17:1731–7.
Zhang F, Dong W, Zeng W, et al. Naringenin prevents TGF-β1 secretion from breast cancer and suppresses pulmonary metastasis by inhibiting PKC activation. Breast Cancer Res. 2016;18:38.
Simon JP, Ivanov IE, Adesnik M, Sabatini DD. The production of post-Golgi vesicles requires a protein kinase C-like molecule, but not its phosphorylating activity. J Cell Biol. 1996;135:355–70.
Sieburth D, Madison JM, Kaplan JM. PKC-1 regulates secretion of neuropeptides. Nat Neurosci. 2007;10:49–57.
Wanger TM, Dewitt S, Collins A, Maitland NJ, Poghosyan Z, Knäuper V. Differential regulation of TROP2 release by PKC isoforms through vesicles and ADAM17. Cell Signal. 2015;27:1325–35.
Siddiqi SA, Mansbach CM 2nd. PKC zeta-mediated phosphorylation controls budding of the pre-chylomicron transport vesicle. J Cell Sci. 2008;121:2327–38.
Takahashi S, Anada M, Kinoshita T, Nishide T, Shibata T. Prospective exploratory study of the relationship between radiation pneumonitis and TGF-β1 in exhaled breath condensate. In Vivo. 2022;36(3):1485–90.
Shelke GV, Yin Y, Jang SC, et al. Endosomal signalling via exosome surface TGFβ-1. J Extracell Vesicles. 2019;8:1650458.
Gopalakrishna R, Anderson WB. Ca2+- and phospholipid-independent activation of protein kinase C by selective oxidative modification of the regulatory domain. Proc Natl Acad Sci USA. 1989;86:6758–62.
Knapp LT, Klann E. Superoxide-induced stimulation of protein kinase C via thiol modification and modulation of zinc content. J Biol Chem. 2000;275:24136–45.
Jobling MF, Mott JD, Finnegan MT, et al. Isoform-specific activation of latent transforming growth factor beta (LTGF-beta) by reactive oxygen species. Radiat Res. 2006;166:839–48.
Ciardiello D, Elez E, Tabernero J, Seoane J. Clinical development of therapies targeting TGFβ: current knowledge and future perspectives. Ann Oncol. 2020;31:1336–49.
Bluwstein A, Kumar N, Léger K, et al. PKC signaling prevents irradiation-induced apoptosis of primary human fibroblasts. Cell Death Dis. 2013;4:e498.
Dhainaut M, Rose SA, Akturk G, et al. Spatial CRISPR genomics identifies regulators of the tumor microenvironment. Cell. 2022;185:1223–39.
Liu X, Wang W, Hu H, et al. Smad3 specific inhibitor, naringenin, decreases the expression of extracellular matrix induced by TGF-beta1 in cultured rat hepatic stellate cells. Pharm Res. 2006;23:82–9.
Lou C, Zhang F, Yang M, et al. Naringenin decreases invasiveness and metastasis by inhibiting TGF-β-induced epithelial to mesenchymal transition in pancreatic cancer cells. PLoS ONE. 2012;7:e50956.
Zhang C, Zeng W, Yao Y, et al. Naringenin ameliorates radiation-induced lung injury by lowering IL-1β level. J Pharmacol Exp Ther. 2018;366:341–8.
We would like to thank all participants enrolled in this study. We also thank Lingtao Jin in Department of Anatomy and Cell Biology, College of Medicine, University of Florida for fruitful discussions.
This work was supported by National Natural Science Foundation of China 81773288 (to F. Z.).
Ethics approval and consent to participate
The experimental protocols of this study were approved by the Animal Care and Use Committee of Institute of Biophysics, Chinese Academy of Science, China (SYXK201707, SYXK2022133), The using of tissues from patients with NSCLC was approved by the ethics committees at Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (NCC2022C-702, from June 8th, 2022), and all experiments were performed in accordance with relevant guidelines and regulations.
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The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Figure S1 EVs promote the differentiation of naïve CD4 + T cells to Tregs. Naïve CD4+T cells were isolated from Foxp3-GFP mice and incubated with anti-CD3/CD28 functional antibody with EVs (500 µg/mL) or TGF-β1 (5 ng/mL) for 72 h followed by flow cytometry analysis. A The differentiation of naïve CD4+ T cells to Treg induced by EVs was detected by quantification of the percentage of CD25+GFP+cells in CD4+ cells. B The percentage of Ki67+ cells in Treg cells was quantified by flow cytometry analysis. C The percentage of Annexin V+ cells in Treg cells was quantified by flow cytometry analysis. ****p < 0.0001. All experiments was analyzed by t test. Figure S2 TGF-β1 is largely associated with EVs in 4T1 cells and tumors after radiation treatment. A 4T1 cells were treated with 8 Gy radiation and cultured for 48 h. EVs were isolated from supernatant and ELISA was performed for detection of TGF-β1. B 4T1 breast cancer tumors carried by C57BL/6 mice were treated with 10 Gy of radiation. Tumors were isolated 14 days after radiation, the tumor tissues were digested and EVs were extracted. Total TGF-β1 and TGF-β1 associated with EVs in the suspension were detected using TGF-β1 ELISA kit. ****p < 0.0001. All experiments was analyzed by 2-way ANOVA. Figure S3 PKC-ζ siRNA effectively inhibits mRNA and protein expression in 4T1 cells. A 4T1 cells were transfected with PKC-ζ siRNA for 24 or 48 h and RNA was isolated for qPCR. B Western blot was performed for detection of PKC-ζ protein expression after PKC-ζ siRNA treatment. Figure S4 3D structures of PKC isoforms. A Structure of the zinc finger motifs of different mouse PKC isoforms adapted from the PKC structure predicted by AlphaFold. The cysteine residues are highlighted in blue. B The PKC structure of different mouse PKC isoforms, with the first zinc finger motif highlighted in cyan while the second highlighted in red. Figure S5 PKCs mRNA relative expression and zinc release in MDA-MB-231 cells. A MDA-MB-231 cells were collected and the mRNA relative expression of PRKCZ and PRKCI to β-Actin was analyzed using qPCR. B Naringenin (Nar, 200 µM) were added to MDA-MB-231 cells for 30 min before different dose of X-Ray (0, 2, 4 and 8 Gray) administration. The relative fluorescence units were measured by the fluorescence microplate reader. *p < 0.05; **p < 0.01. Data were analyzed by t-test. Figure S6 RT induced the entry of NFkB into nuclear, which was modulated by naringenin. 4T1 cells were treated with 8 Gy of X-Ray (RT) and 200uM of Naringenin (RT + Nar) for 2 h. Total proteins were extracted and isolated into cytoplasmic and nuclear sections. Western blot was performed for detection of NFkB protein expression in nuclear section with β-Actin as its internal reference after different treatment.
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Zhang, F., Zheng, Z., Wang, L. et al. PKC-ζ mediated reduction of the extracellular vesicles-associated TGF-β1 overcomes radiotherapy resistance in breast cancer. Breast Cancer Res 25, 38 (2023). https://doi.org/10.1186/s13058-023-01641-4