[Corrigendum] Increased infiltration of macrophages to radioresistant lung cancer cells contributes to the development of the additional resistance of tumor cells to the cytotoxic effects of NK cells.

Subsequently to the publication of the above paper, the authors have realized that the western blots featured in Fig. 5B were inadvertently copied across from Fig. 4B owing to an error made during the figure compilation process. The corrected version of Fig. 5 is featured on the next page, showing the correct data for the western blot analysis of the programmed death receptor ligand 1 level in radioresistant lung cancer cells under the specified experimental conditions. Note that these changes do not affect the interpretation of the data or the conclusions reported in this paper, and all the authors agree to this correction. The authors apologize to the Editor and to the readership of the Journal for any inconvenience caused. [the original article was published in International Journal of Oncology 53: 317-328, 2018; DOI: 10.3892/ijo.2018.4394].

Glucocorticoid receptor upregulation increases radioresistance and triggers androgen independence of prostate cancer.

BACKGROUND: Despite the overall success of radiotherapy, a significant number of patients develop radioresistance, which leads to local regional recurrence and distant metastasis. We studied whether repeated radiation treatment promotes androgen-independent survival of prostate cancer (PCa) cells and their metastatic potential. We also studied whether glucocorticoid receptor (GR) increase in radioresistant cells is associated with acquisition of these aggressive characteristics. METHODS: Radioresistant LNCaP (LNCaPR18) and C4-2 (C4-2R26) PCa sublines were developed by repeated radiation treatments of parental cells. Levels and activations of androgen receptor (AR) and GR in radioresistant PCa cells and respective parental cells were investigated in quantitative real-time polymerase chain reaction/Western blot analyses and immunofluorescence staining. Androgen-independent survival of radioresistant cells was tested in in vitro cell growth assays and the castration-resistant survival of these cell-derived tumors were investigated in mouse xenografts. RESULTS: Higher GR levels, but lower AR levels were detected in radioresistant cells than in parental cells. Radiation-induced GR upregulation was associated with increased intracellular cyclic adenosine monophosphate. As a consequence of GR activation, LNCaPR18 cells survived well in an androgen-depleted culture condition while parental cells could not. Results of in vivo mouse studies showed survival of LNCaPR18 cell-derived tumors in castrated mice while parental cell-derived tumors regressed. The growth of LNCaPR18 cell-derived tumors in castrated mice was impaired when treated with the anti-GR agent mifepristone. In experiments with C4-2/C4-2R26 cell sets, GR activation in C4-2R26 cells increased their metastatic potential. CONCLUSION: GR activation in radioresistant cells mediates androgen independence and facilitates PCa progression.

Radiation-induced glucocorticoid receptor promotes CD44+ prostate cancer stem cell growth through activation of SGK1-Wnt/ß-catenin signaling.

We observed cancer stem cell (CSC) population increase in radioresistant LNCaP (LNCaPR18) and C4-2 (C4-2R26) prostate cancer (PCa) cells compared with respective parental cells. Since the CD44 level increase was most significant in radioresistant PCa cells compared with parental cells among CSC markers tested, we isolated the CD44+ population from LNCaP/LNCaPR18 and C4-2/C4-2R26 cell sets via the immunomagnetic separation method and used them as CSC sources. We detected lower AR level, but higher glucocorticoid receptor (GR) level in CD44+ CSCs than CD44- non-CSCs. Higher GR level in CD44+ CSCs than CD44- cells was also detected when cells were isolated from mouse tumor tissues of LNCaPR18 cell and C4-2R26 cell-derived human xenografts and grown in culture. We then found blocking the GR signaling by adding the anti-GR agent mifepristone into the cell culture inhibited the CD44+ CSC growth while the addition of the anti-AR agent enzalutamide enhanced the CSC growth. In xenograft mouse studies in which tumors were developed from the injection of CD44+ CSCs of LNCaPR18 or C4-2R26 cell lines, retarded tumor growth in mifepristone-injected mice was observed compared with vehicle-treated mice. We next discovered the GR regulation of Wnt/ß-catenin signaling pathway. We further found that the serum/glucocorticoid regulated kinase 1 (SGK1) is the GR downstream molecule that mediates Wnt/ß-catenin signaling activation. Therefore, inhibition of either SGK1 or Wnt/ß-catenin signaling impaired the in vitro CD44+ CSC growth. From these results, we suggest that blocking GR signaling or its downstream SGK1-Wnt/ß-catenin signaling axis may suppress the radiation-induced CSC increase in PCa. KEY MESSAGES: Higher CSC population exists in radioresistant PCa cells than parental cells. Higher GR levels (and lower AR level) in CD44+ CSCs than CD44- non-CSCs. Use of anti-GR agent blocked the growth of CD44+ CSCs in in vitro/in vivo tests. GR downstream SGK1-Wnt/ß-catenin signaling axis mediates the CSC increase. Targeting this signaling axis may enhance the radiotherapy efficacy in treating PCa.

IL-6 signaling contributes to radioresistance of prostate cancer through key DNA repair-associated molecules ATM, ATR, and BRCA 1/2.

PURPOSE: To study an association between IL-6 signaling and resistance to radiotherapy of prostate cancer (PCa) and explore the molecular mechanisms involved. METHODS: IL-6 expressing C4-2 and CWR22Rv1 (C4-2IL-6/CWRIL-6) and vector control (C4-2vec/CWRvec) cell lines were developed. Radiation-sensitivities of these cells were compared in clonogenic assay, Comet assay, and γH2AX staining. In xenograft animal studies, radiation-sensitivity of C4-2IL-6 cell-derived tumors vs. C4-2vec cell-derived tumors was investigated. To reveal IL-6 downstream molecules involved in DNA repair after radiation, qPCR and Western blot analyses as well as immunofluorescence staining were performed. Transcriptional control of IL-6 on ATM and ATR molecules was also investigated. RESULTS: We found C4-2IL-6 and CWRIL-6 cells survived better than their vector control cells after irradiation, and animal studies confirmed such in vitro results. We discovered that DNA repair-related molecules such as ATM, ATR, BRCA1, and BRCA2 were significantly upregulated in irradiated IL-6 expressing cells compared with vector control cells. We further defined that IL-6 signaling regulated cellular expressions of ATM and ATR at the transcriptional level through the activation of Stat3 signaling pathway. CONCLUSIONS: IL-6 leads to PCa resistance to radiation through upregulation of DNA repair associated molecules ATM, ATR, BRCA1, and BRCA2.

NFκB and TNFα as individual key molecules associated with the cisplatin-resistance and radioresistance of lung cancer.

Cisplatin-resistant (A549CisR and H292CisR) and radioresistant (A549R26 and H292R22) sub-line non-small cell lung cancer (NSCLC) cells were developed in our lab by long term treatment of parental cells with cisplatin or radiation. Our data showed no cross-resistance between these two sets of cell lines, indicating that molecular mechanisms of developing each resistance may be different. Using these sub-line cells, we sought to reveal the most significantly up-regulated molecules in cisplatin-resistant and radioresistant lung cancer cells, compared with parental cells. In qPCR analyses of screening DNA repair and cell survival-associated molecules, we identified NFκB and TNFα as the most significantly up-regulated molecules in cisplatin-resistant and radioresistant lung cancer cells, respectively, compared with parental cells. Western blot analysis of parental vs. resistant cells and the IHC staining of tumor tissues of A549P, A549CisR, and A549R26 cell-derived xenografts in mice confirmed such results. Next, studies using specific inhibitors of NFκB and TNFα and experiments using NFκB and TNFα-knocked down cells showed that inhibition or knockdown of NFκB overcame cisplatin-resistance, while inhibition or knockdown of TNFα increased radiosensitivity of radioresistant lung cancer cells. Therefore, these two molecules may be used as markers of the prognosis/diagnosis of individual resistance development during lung cancer treatment.

RETRACTED: Neuroendocrine differentiation contributes to radioresistance development and metastatic potential increase in non-small cell lung cancer.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the authors and their institute. The BBA Editor-in-Chief has agreed to retract the paper. In this paper, there were two errors identified to the journal by the authors: The first error was in Western blot gel band images of Fig. 4A (p-MAPK, MAPK, p-Erk, and Stat3) and the 8 gel band images of Fig. 4G. The second error was in the cell culture images of Figures 3F, 3J, and 4E. The authors state that these errors were due to uploading mistakes in the preparation of the manuscript. The authors apologize for these errors and any inconvenience caused. The Editor-in-Chief initially agreed to retract the paper based on the identification of these two errors. Readers are able to see further discussion of the paper on the PubPeer site here: https://pubpeer.com/publications/569EB2CE7A7335D7F3F8F3FF310936

Increased infiltration of macrophages to radioresistant lung cancer cells contributes to the development of the additional resistance of tumor cells to the cytotoxic effects of NK cells.

In this study, in order to investigate the effects of increased macrophage infiltration to radioresistant lung tumors in regulating natural killer (NK) cell-mediated immunity, we examined whether the treatment of radioresistant cells with conditioned medium (CM) from phorbol myristate acetate (PMA)/interleukin (IL)-4 treated THP-1 cells (used as a tumor-associated macrophage source) leads to the development of the additional resistance of tumor cells to NK cell cytotoxicity. We found that the susceptibility of THP-1 CM-treated radioresistant cells to NK cell cytotoxicity was decreased compared to the non-treated cells. In addition, it was found that such a decreased susceptibility was associated with increased programmed death receptor ligand 1 (PD-L1) and decreased natural killer group 2D (NKG2D) ligand levels in tumor cells. We further discovered that the THP-1 cells secreted a high level of IL-6, and that blocking IL-6 action by the addition of a neutralizing antibody (Ab) for IL-6 into the THP-1 CM decreased the resistance of THP-1 CM-treated radioresistant cells to NK cell cytotoxicity. Moreover, we discovered that MEK/Erk was the most critical IL-6 downstream signaling pathway in triggering the THP-1 CM effect; thus, the addition of MEK/Erk inhibitor to THP-1 CM enhanced the susceptibility of the THP-1 CM-treated radioresistant cells to NK cell cytotoxicity. On the whole, the findings of this study suggest the existence of a malignant loop characterized by increased macrophage infiltration into radioresistant cells which, in turn, promotes the development of the additional resistance of these cells to NK cell cytotoxicity.

In vitro-induced M2 type macrophages induces the resistance of prostate cancer cells to cytotoxic action of NK cells.

Previous reports, including our experimental results, showed that macrophages migrate to prostate cancer (PCa) cells. We tested whether the migrated macrophages affect the susceptibility of castration-resistant PCa (CRPC) cells to cytotoxic actions of natural killer (NK) cells. We found treatment of tumor cells with the conditioned media (CM) of the PMA/IL-4 treated THP-1 cells (M2 type macrophages) (THP-1 CM) decreased the susceptibility of tumor cells to NK cell cytotoxicity, as a result of increased programmed death receptor ligand 1 (PD-L1) and decreased NK group 2D (NKG2D) ligands in CRPC cells. Meanwhile, the decreased susceptibility of tumor cells was also detected when NK cells were treated with THP-1 CM and used in NK cell cytotoxicity tests. Therefore, we observed higher resistance of CRPC cells when both tumor and NK cells were treated with THP-1 CM than when tumor cells or NK cells were individually treated. We further discovered that the PMA/IL-4 treated THP-1 cells secrete a high level of IL-6, so blocking the IL-6 action significantly decreased the PD-L1 level while recovering the NKG2D ligands, thus increasing the susceptibility of CRPC cells to NK cell action. Moreover, we discovered that JAK-Stat3 is the most critical IL-6 downstream signaling in triggering the THP-1 CM effect. Consequently, we found the susceptibility of CRPC cells to NK cells was increased when either JAK or Stat 3 inhibitor was added when tumor cells were treated with THP-1 CM, and that the best effect was observed when the JAK inhibitor and PD-L1 Ab were added together.

Adipocytes affect castration-resistant prostate cancer cells to develop the resistance to cytotoxic action of NK cells with alterations of PD-L1/NKG2D ligand levels in tumor cells.

BACKGROUND: Obesity affects prostate cancer (PCa) progression, and the periprostatic adipose tissue adjacent to the prostate is considered a driving force of disease progression. Adipocytes are the main cell population in adipose tissues and their paracrine role contributes to PCa progression, however its implication in modulating immune reactions remains largely unknown. We investigated the adipocyte role in controlling the susceptibility of castration-resistant PCa (CRPC) cells to the cytotoxic action of natural killer (NK) cells. METHODS: Using primary NK cells as the NK cell source, NK cell cytotoxicities to CRPC cells, either control media treated or adipocyte-conditioned media (CM) treated, were tested in lactate dehydrogenase (LDH) release-based assays. The levels of programmed death receptor ligand (PD-L1) and NK group 2D (NKG2D) ligands in adipocyte CM-treated CRPC cells were analyzed in qPCR analyses. Effects of blocking adipocyte action on altering PD-L1/NKG2D ligand levels and the susceptibility of CRPC cells to NK cell cytotoxicity were investigated. RESULTS: We found NK cell cytotoxicity to CRPC cells decreases when tumor cells are treated with adipocyte CM associated with PD-L1 and NKG2D ligand level alterations. Further, we discovered that the JAK/Stat3 signaling pathway was responsible for the adipocyte CM effect. Two adipokine molecules, IL-6 and leptin, were shown to be important in activation of the JAK/Stat3 signaling in CRPC cells to modulate the PD-L1/NKG2D ligand level alteration. Adding the inhibitors of JAK/Stat3 signaling or neutralizing antibodies of IL-6 or leptin increased the susceptibility of CRPC cells to NK cell action. CONCLUSIONS: Blocking the adipocyte effect by inhibiting the IL-6/leptin-JAK/Stat3 signaling axis may enhance NK cell mediated immunity to CRPC cells and this strategy may help to develop future therapeutics to treat obese PCa patients.

FASN-TGF-ß1-PD-L1 axis contributes to the development of resistance to NK cell cytotoxicity of cisplatin-resistant lung cancer cells.

Cisplatin remains the most effective therapy for non-small cell lung cancer (NSCLC). We previously have found cisplatin-resistant lung cancer cells (A549CisR and H157CisR) were more resistant to natural killer (NK) cell-mediated cytotoxicity than parental cells. We also discovered that fatty acid synthase (FASN) levels in cisplatin-resistant cells were significantly higher than in parental cells. To reveal whether a link exists between the up-regulated FASN levels and higher resistance to NK cell cytotoxicity, we performed inhibition studies using a FASN inhibitor and applied the FASN knockdown approach. In both approaches, we found that the FASN inhibition/knockdown significantly increased the susceptibility of cisplatin-resistant cells to NK cell cytotoxicity. We further found such decreased susceptibility was associated with an increased programmed death receptor ligand (PD-L1) level in cisplatin-resistant cells. In mechanisms studies, TGF-ß1 was found to be the FASN downstream signaling molecule that was responsible for modulating the PD-L1 levels in cisplatin-resistant cells. Accordingly, TGF-ß1 inhibition resulted in significantly increased susceptibility of cisplatin-resistant cells to NK cell cytotoxicity. We suggest that the inhibition of FASN-TGFß1-PD-L1 axis may improve the efficacy of immunotherapy in treating cisplatin-resistant lung cancer.

Inhibition of IL-6-JAK/Stat3 signaling in castration-resistant prostate cancer cells enhances the NK cell-mediated cytotoxicity via alteration of PD-L1/NKG2D ligand levels.

To investigate whether IL-6 signaling affects the susceptibility of castration-resistant prostate cancer (CRPC) cells to cytotoxic action of natural killer (NK) cells, CRPC cell lines (having different IL-6 levels) were developed by lentiviral transduction. While observing no secreted IL-6 level in parental C4-2 and CWR22Rv1 cells, we found the IL-6 expression/secretion in these cells was induced after the transduction process and the IL-6 level difference in C4-2siIL-6/sc and CWR22siIL-6/sc cell CRPC cell sets could be detected. We then found that IL-6-knockdown cells were more susceptible to NK cell cytotoxicity than control cells due to lowered programmed death receptor ligand 1 (PD-L1) and increased NK group 2D (NKG2D) ligand levels. In animal studies, to concur with the in vitro results, we found that IL-6-expressing cell-derived tumors were more resistant to NK cell action than the tumors of IL-6-knockdown cells. Further, we discovered that JAK-Stat3 is the most critical IL-6 downstream signaling that modulates PD-L1/NKG2D ligand levels in CRPC cells. Furthermore, inhibition of the JAK or Stat3 signaling effectively increased the susceptibility of C4-2sc and CWRsc cells to NK cell cytotoxicity. We observed the most effective cytotoxicity when the PD-L1 Ab and JAK inhibitor (or Stat 3 inhibitor) were used together. These results suggest that the strategy of targeting IL-6 signaling (or its downstream signaling) may enhance the NK cell-mediated immune action to CRPC tumors, thus yielding clinical implications in developing future immunotherapeutics of exploiting this strategy to treat patients with CRPC.

Radiation alters PD-L1/NKG2D ligand levels in lung cancer cells and leads to immune escape from NK cell cytotoxicity via IL-6-MEK/Erk signaling pathway.

We investigated whether radiation influences the susceptibility of non-small cell lung cancer (NSCLC) cells to NK cell mediated cytotoxicity. We found radiation treatment increased expression of programmed cell death ligand 1 (PD-L1), but decreased NK group 2, member D (NKG2D) ligand expressions in A549 and H157 NSCLC cells. Both types of changes would have protected tumor cells from the cytotoxic action of NK cells. Consistently, we detected similar alteration in these molecules in radioresistant A549R26-1 and H157R24-1 subline cells. Higher PD-L1 level was also observed in tumors of A549R26-1 cell-derived xenografts than tumors of parental A549 (A549P) cell-derived xenografts. Accordingly, we found radioresistant cells were more resistant to the cytotoxic action of NK cells than parental cells, and such resistance was decreased when neutralizing antibody (Ab) of PD-L1 was added to the radioresistant cell/NK cell co-cultures. In mechanism studies, we found that IL-6-MEK/Erk signaling contributed most significantly to the up-regulation of PD-L1/down-regulation of NKG2D ligands in radioresistant cells. The addition of the MEK/Erk inhibitor increased the susceptibility of A549R26-1 and H157R24-1 cells to NK-cell cytotoxicity while no significant effect was observed in parental cells. Moreover, we detected enhanced NK-cell cytotoxicity to radioresistant cells when PD-L1 Ab and MEK/Erk inhibitor were added together to co-cultures of tumor/NK cells compared to when PD-L1 Ab was used alone. We suggest that combined use of PD-L1 Ab and MEK/Erk inhibitor may offer better therapeutic benefits than PD-L1 Ab alone to treat NSCLC patients who are receiving radiotherapy or who are at the radioresistant stage.

Enhancing NK cell-mediated cytotoxicity to cisplatin-resistant lung cancer cells via MEK/Erk signaling inhibition.

Major progress has been made clinically in inhibiting the programmed death receptor 1 (PD-1)/PD-L1 interaction to enhance T cell-mediated immune function, yet the effectiveness of anti-PD-L1/PD-1 agents in enhancing natural killer (NK) cell's function remains largely unknown. Susceptibilities of cisplatin-resistant A549CisR and H157CisR cells vs. parental cells to the cytotoxic action of NK cells were examined. We found cisplatin-resistant cells more resistant to NK cell cytotoxicity than parental cells. There were constitutively higher expressions of PD-L1 in A549CisR and H157CisR cells than in parental cells in vitro, as well as in H157CisR cell-derived tumors than H157P cell-derived tumors. In contrast, we observed that the expression of PD-1 in NK cells was induced after co-culture with cisplatin-resistant cells. We also observed increased susceptibility of cisplatin-resistant cells to NK cell cytotoxicity when neutralizing antibody of PD-1 or PD-L1 was added. Further, we found that the NK group 2, member D (NKG2D) ligand levels were lower in A549CisR and H157CisR cells than in parental cells. Meanwhile, we discovered that the MEK/Erk signaling pathway played a significant role in this regulation, and the addition of a MEK/Erk pathway inhibitor significantly enhanced the PD-L1 Ab effect in enhancing NK cell cytotoxicity to cisplatin-resistant cells.

Simultaneous targeting of ATM and Mcl-1 increases cisplatin sensitivity of cisplatin-resistant non-small cell lung cancer.

Development of cisplatin-resistance is an obstacle in non-small cell lung cancer (NSCLC) therapeutics. To investigate which molecules are associated with cisplatin-resistance, we analyzed expression profiles of several DNA repair and anti-apoptosis associated molecules in parental (A549P and H157P) and cisplatin-resistant (A549CisR and H157CisR) NSCLC cells. We detected constitutively upregulated nuclear ATM and cytosolic Mcl-1 molcules in cisplatin-resistant cells compared with parental cells. Increased levels of phosphorylated ATM (p-ATM) and its downstream molecules, CHK2, p-CHK2, p-53, and p-p53 were also detected in cisplatin-resistant cells, suggesting an activation of ATM signaling in these cells. Upon inhibition of ATM and Mcl-1 expression/activity using specific inhibitors of ATM and/or Mcl-1, we found significantly enhanced cisplatin-cytotoxicity and increased apoptosis of A549CisR cells after cisplatin treatment. Several A549CisR-derived cell lines, including ATM knocked down (A549CisR-siATM), Mcl-1 knocked down (A549CisR-shMcl1), ATM/Mcl-1 double knocked down (A549CisR-siATM/shMcl1) as well as scramble control (A549CisR-sc), were then developed. Higher cisplatin-cytotoxicity and increased apoptosis were observed in A549CisR-siATM, A549CisR-shMcl1, and A549CisR-siATM/shMcl1 cells compared with A549CisR-sc cells, and the most significant effect was shown in A549CisR-siATM/shMcl1 cells. In in vivo mice studies using subcutaneous xenograft mouse models developed with A549CisR-sc and A549CisR-siATM/shMcl1 cells, significant tumor regression in A549CisR-siATM/shMcl1 cells-derived xenografts was observed after cisplatin injection, but not in A549CisR-sc cells-derived xenografts. Finally, inhibitor studies revealed activation of Erk signaling pathway was most important in upregulation of ATM and Mcl-1 molcules in cisplatin-resistant cells. These studies suggest that simultaneous blocking of ATM/Mcl-1 molcules or downstream Erk signaling may recover the cisplatin-resistance of lung cancer.

IL-6 Mediates Macrophage Infiltration after Irradiation via Up-regulation of CCL2/CCL5 in Non-small Cell Lung Cancer.

Radiotherapy is effective in reducing primary tumors, however, it may enhance macrophage infiltration to tumor sites, accelerating tumor progression in several ways. We investigated whether radiation can increase macrophage infiltration into non-small cell lung carcinoma (NSCLC) cells. Analysis of in vitro macrophage (differentiated THP-1 cells) migration to either nonirradiated or irradiated tumor cells showed increased migration to the irradiated tumor cells. Because the IL-6 levels in A549 and H157 cells were significantly increased after irradiation, we then investigated whether this increased IL-6 level contributes to radiation-induced macrophage migration. Radiation-induced macrophage infiltration was reduced when IL-6 was knocked down in tumor cells, indicating a positive IL-6 role in this process. To validate this in vitro result, an orthotopic mouse model was developed using a luciferase-tagged H157siIL-6/scramble control (sc) cell set. After tumors developed, the lungs were irradiated, and infiltration of endogenous macrophages and tail-vein injected fluorescent macrophages to tumor sites was investigated. In both groups, increased macrophage infiltration was observed in H157sc cell-derived xenografts compared to H157siIL-6 cell-derived xenografts, confirming the positive IL-6 role in the radiation-induced macrophage infiltration process. In mechanistic dissection studies, radiation-induced up-regulation of CCL2 and CCL5 by IL-6 was detected, and blocking the action of CCL2/CCL5 molecules significantly reduced the number of migrated macrophages to tumor cells after irradiation. These results demonstrate that targeting the IL-6 signaling or CCL2/CCL5 molecules in combination with conventional radiotherapy potentially blocks undesired radiation-induced macrophage infiltration.