Toripalimab plus chemotherapy as second-line treatment in previously EGFR-TKI treated patients with EGFR-mutant-advanced NSCLC: a multicenter phase-II trial.

This multicenter phase-II trial aimed to investigate the efficacy, safety, and predictive biomarkers of toripalimab plus chemotherapy as second-line treatment in patients with EGFR-mutant-advanced NSCLC. Patients who failed from first-line EGFR-TKIs and did not harbor T790M mutation were enrolled. Toripalimab plus carboplatin and pemetrexed were administrated every three weeks for up to six cycles, followed by the maintenance of toripalimab and pemetrexed. The primary endpoint was objective-response rate (ORR). Integrated biomarker analysis of PD-L1 expression, tumor mutational burden (TMB), CD8 + tumor-infiltrating lymphocyte (TIL) density, whole-exome, and transcriptome sequencing on tumor biopsies were also conducted. Forty patients were enrolled with an overall ORR of 50.0% and disease-control rate (DCR) of 87.5%. The median progression free survival (PFS) and overall survival were 7.0 and 23.5 months, respectively. The most common treatment-related adverse effects were leukopenia, neutropenia, anemia, ALT/AST elevation, and nausea. Biomarker analysis showed that none of PD-L1 expression, TMB level, and CD8 + TIL density could serve as a predictive biomarker. Integrated analysis of whole-exome and transcriptome sequencing data revealed that patients with DSPP mutation had a decreased M2 macrophage infiltration and associated with longer PFS than those of wild type. Toripalimab plus chemotherapy showed a promising anti-tumor activity with acceptable safety profiles as the second-line setting in patients with EGFR-mutant NSCLC. DSPP mutation might serve as a potential biomarker for this combination. A phase-III trial to compare toripalimab versus placebo in combination with chemotherapy in this setting is ongoing (NCT03924050).

Effects of DKK1 overexpression on bone metastasis of SBC-3 cells.

Among all malignancies, lung cancer is the leading cause of cancer-related deaths in China. Bone metastasis is one of the most common complications and one of the most important factors affecting the prognosis of lung cancer patients, which resulting in very poor therapeutic effects. Previously, we have demonstrated that the expression levels of Dickkopf1 (DKK1), a protein involved in cell regulation and proliferation, was dramatically higher in cells that have a tendency to metastasize and invade the bone tissue (SBC-5 cells) compared with cells that do not (SBC-3 cells). Downregulation of DKK1 in SBC-5 cells inhibited cell malignancy in vitro, and the formation of bone metastasis in vivo. However, whether upregulating DKK1 would be sufficient to induce aggressive tumor behavior (proliferation, migration, invasion and metastasis) in SBC-3 cells remained to be investigated. The present study aimed to examine the role of DKK1 in SBC-3 cells, as well as to investigate the SBC-3 ability to metastasize and invade the bone tissue. The results demonstrated that upregulation of DKK1 in SBC-3 cells enhanced cell proliferation, colony formation, cell migration and invasion in vitro, as well as bone metastasis in vivo. These results indicate that DKK1 may be an important regulator in the development of small cell lung cancer (SCLC), and targeting DKK1 may be an effective method for preventing and/or treating skeletal metastases in SCLC cases.

The effect of RCAN1 on the biological behaviors of small cell lung cancer.

Bone is the third most common site of cancer metastasis. In total, 30%-40% of lung cancer cases can develop skeletal metastasis for which no effective therapy in clinic is available. RCAN1 (regulator of calcineurin 1) is an important regulator in angiogenesis which is vital to tumor growth. In this study, we investigated the changes of biological behaviors in SBC-5 and SBC-3 cells after the RCAN1 expression level was changed. Briefly, overexpression of RCAN1 significantly attenuated their malignancy, including decreased ability of proliferation, colony formation, migration, invasion, and bone adherence. Furthermore, the cell cycle progression was impeded. Although the opposite changes were observed in SBC-3 cells after the RCAN1 expression was suppressed by RNA interference, the apoptosis rate was not affected by the expression level of RCAN1 in these cells. So, our research revealed that RCAN1 was involved in the development of small cell lung cancer, and it might be a cancer-inhibiting gene for the formation of bone metastases in small cell lung cancer.

The Biological Effects of Dickkopf1 on Small Cell Lung Cancer Cells and Bone Metastasis.

The bone is among the most common sites of metastasis in patients with lung cancer. Over 30%-40% of lung cancers can develop bone metastasis, and no effective therapeutic methods exist in clinic cases. Wnt/ß-catenin signaling and Dickkopf1 (DKK1) play important roles in the progression of lung cancer, which preferentially metastasizes to the skeleton. However, the role of DKK1 in osteotropism of small cell lung cancer (SCLC) remains to be elucidated. This study aimed to define the role of DKK1 in SCLC bone metastasis and investigate the underlying mechanisms. Our results demonstrated that the expression level of DKK1 was dramatically higher in bone metastatic SCLC cells (SBC-5 cell line) compared with that in cells without bone metastatic ability (SBC-3 cell line). Therefore, we hypothesized that DKK1 was involved in the bone metastasis of SCLC. We then suppressed the DKK1 expression in SBC-5 cells by RNAi and found that downregulation of DKK1 can inhibit cell proliferation, colony formation, cell migration, and invasion, but increase the apoptosis rate. Downregulation of DKK1 did not affect the cell cycle progression of SBC-5 cells in vitro. In vivo, downregulated DKK1 in SBC-5 cells resulted in attenuated bone metastasis. These results indicated that DKK1 may be an important regulator in bone metastases of SCLC, and targeting DKK1 may be an effective method to prevent and treat skeleton metastases in SCLC cases.

Downregulation of CXCR4 by SDF-KDEL in SBC-5 cells inhibits their migration in vitro and organ metastasis in vivo.

Metastasis is the principal cause of morbidity and mortality in cancer patients. The master genes that govern organ-selective metastasis remain elusive. We compared the expression levels of C-X-C chemokine receptor type 4 (CXCR4) in the human small cell lung cancer (SCLC) cells, SBC-5 and SBC-3, by flow cytometric analysis and found that CXCR4 was expressed at markedly higher levels in the SBC-5 cells which can produce multiple organ metastasis, particularly bone metastasis compared to the SBC-3 cells. Stromal-derived-factor-1 (SDF-1)-CXCR4 has been shown to regulate cell migration and metastasis in a various types of cancer; however, the roles of SDF-1-CXCR4 in the organ-selective metastasis of SCLC in vivo remain to be elucidated. Thus, in this study, we constructed a phenotype of SBC-5 cells in which CXCR4 was knocked out using the intrakine strategy and found that the downregulation of CXCR4 inhibited cell migration and invasion, but did not affect cell proliferation or apoptosis in vitro. In in vivo experiments, the knockout of CXCR4 suppressed the development of metastastic lesions in the lungs, liver and bone, but did not decrease metastasis to the kidneys. Our data demonstrate that CXCR4 is a candidate gene involved in the development of metastastic lesions in specific organs, such as the lungs, bone and liver, which can secrete high concentrations of SDF-1, the sole ligand of CXCR4. Thus, CXCR4 may prove to be a promising target for the prevention and effective treatment of metastastic lesions due to SCLC.

Epithelial-mesenchymal transition contributes to docetaxel resistance in human non-small cell lung cancer.

Lung cancer is an aggressive malignancy with high morbidity and mortality. Chemotherapy has always been the principal treatment measure, but its acquired resistance becomes a critical problem. In the current study, we established a new docetaxel-resistant human non-small lung cancer (NSCLC) cell line A549/Docetaxel. The resistance index (RI) of A549/Docetaxel cells and A549 induced by TGF-ß to docetaxel were 8.91 and 11.5, respectively. Compared to the parental A549 cells, the multiplication time of A549/Docetaxel was prolonged, the proportion of the cell cycle in the S phase decreased while that in the G1 phase increased, and apoptotic rate was much lower. The morphology of the resistant cells eventuated epithelial-mesenchymal transition (EMT), which was confirmed by the higher expression of fibronectin, vimentin (mesenchymal markers), and lower expression of E-cadherin (epithelial marker) at mRNA and proteins levels. Furthermore, the representative markers for docetaxel resistance were examined, including ABCB1 (MDR1), Bcl-2, Bax, and tubulin, to figure out the mechanisms of the resistance of A549/Docetaxel. In summary, we have established a typical docetaxel-resistant human NSCLC cell line A549/Docetaxel, and it was suggested that the multidrug resistance of A549/Docetaxel was related to EMT.

MGr1-Ag promotes invasion and bone metastasis of small-cell lung cancer in vitro and in vivo.

Bone metastasis of small-cell lung cancer (SCLC) usually occurs early in the progression of the disease. However, the molecular mechanism underlying bone metastasis is largely unknown. MGr1-Ag, a multifunction protein, has been suggested to play important roles in cell growth, differentiation and migration. In our present study, MGr1-Ag was found to be highly expressed in bone-metastatic SCLC cells (SBC-5 cell line) as compared with the expression in cells without bone-metastatic ability (SBC-3 cell line). Therefore, we hypothesized that MGr1-Ag is involved in bone metastasis of SCLC. Using a sense vector to upregulate MGr1-Ag expression in SBC-3 cells, we found that forced overexpression of MGr1-Ag enhanced cell invasion and migration in vitro and promoted bone metastases in vivo. Furthermore, specific siRNA-induced knockdown of MGr1-Ag expression in SBC-5 cells suppressed the potential of cell invasion and migration in vitro and dramatically decreased the number and sites of bone metastasis in vivo. We also found that MGr1-Ag induced SCLC cells to undergo epithelial-mesenchymal transition (EMT), as demonstrated by cell morphological changes, decreased expression of epithelial markers and increased expression of mesenchymal markers. Taken together, we conclude that MGr1-Ag promotes SCLC cell invasion and bone metastasis in vitro and in vivo, and that this is partially mediated via the EMT pathway.

Zinc finger E-box binding homeobox 1 promotes invasion and bone metastasis of small cell lung cancer in vitro and in vivo.

Bone is one of the most frequent targets of small cell lung cancer (SCLC) metastasis and is closely associated with a poor prognosis, but the specific cellular gene alterations responsible for SCLC with bone metastasis are unclear. Zinc finger E-box binding homeobox 1 (ZEB1) as an E-box transcriptional repressor has been suggested that an important inducer of the epithelial-mesenchymal transition (EMT) and a promoter of tumor metastasis in colon, breast and lung cancers. However, the relationship between ZEB1 and SCLC with bone metastasis is unclear. In this study, ZEB1 was found to be highly expressed in bone-metastatic SCLC tissues and cell lines as compared with those that were non-metastatic (P < 0.05). Using a lentivirus RNA interference technique to knockdown ZEB1 expression in bone-metastatic SCLC cells (SBC-5 cell line), we found that ZEB1 siRNA could inhibit the invasive and migratory ability and decrease parathyroid hormone-related protein expression, as determined by invasion assays and enzyme-linked immunosorbent assays. Besides, ZEB1 siRNA significantly inhibited the bone metastasis of SBC-5 cells in vivo. Furthermore, overexpression of ZEB1 in SBC-3 cells, which demonstrate promoted bone-metastatic potential, dramatically promoted their invasive and migratory ability and parathyroid hormone-related protein expression as well as increased the number and sites of bone metastases in vivo compare to the control group. We also found that SBC-3 cells underwent EMT, as indicated by decreased epithelial markers and increased mesenchymal marker expression. Taken together, these results indicate that ZEB1 promoted the invasive ability and bone metastasis of SCLC cells, and that this was partially mediated via the EMT pathway.

S100B protein as a possible participant in the brain metastasis of NSCLC.

Brain metastasis is a frequent occurrence in lung cancer, especially non-small cell lung cancer (NSCLC), the prognosis for NSCLC with brain metastasis is very poor. Our previous study found high S100B expression in the brain-specific metastatic NSCLC line PC14/B, suggested S100B is closely correlated with brain metastasis in NSCLC. However, the details have not yet been revealed. The aim of this study was to investigate the correlation between S100B and brain metastasis in NSCLC and to study the effects of S100B on non-brain metastatic NSCLC line PC14. We investigated serum S100B levels in 30 newly diagnosed NSCLC patients (15 with brain metastasis and 15 without brain metastasis) using enzyme-linked immunosorbent assay. Results showed that serum S100B levels were significant higher in NSCLC patients with brain metastasis compared to those without brain metastasis (P<0.01). We constructed the full-length S100B expression vector and transfected into PC14 cells. MTT and flow cytometric analysis showed that S100B transfection promoted cell proliferation and inhibited cell apoptosis (P<0.05). Transwell migration and invasion assays indicated that S100B transfection promoted cell invasion and cell migration compared to control cells transfected with empty vector alone (P<0.01). These results suggested that S100B could be involved in the development of brain metastasis in NSCLC.

Lentivirus-mediated siRNA interference targeting SGO-1 inhibits human NSCLC cell growth.

Aneuploid is recognized as a hallmark of cancer and is caused by chromosome mis-segregation during mitosis. Recent studies have identified shugoshin (SGO-1) as an important player in proper chromosome segregation and the involvement in tumorigenesis. But, little is known about how SGO-1 is involved. The aim of this study was to obtain information about the role of SGO-1 in human lung cancer cells. In our study, real-time PCR and western blotting assay were performed to detect the expression of SGO-1 in nonsmall cell lung cancer (NSCLC) cell lines. Then, we successfully constructed a lentivirus vector mediating RNAi targeting of SGO-1 (LV-SGO-1 siRNA) and proved that it can suppress the expression of SGO-1 gene in H1299 cells. We found that SGO-1 was highly expressed in NSCLC cancer cell lines. RNA interference of SGO-1 by the LV-SGO-1 siRNA construct significantly decreased SGO-1 protein expression and inhibited the growth and ability of forming colonies of H1299 cells with more cells arrested in G2/M phase, but lentivirus vector control had no effect on H1299 cells. Moreover, suppression of SGO-1 by LV-SGO-1 siRNA increased the apoptosis of H1299 cells with down-regulated Bcl-2 and up-regulated Bax. SGO-1 may provide a novel therapeutic target for the treatment of lung cancer since it inhibits the growth and increases the apoptosis of lung cancer cells.

S100B promotes the proliferation, migration and invasion of specific brain metastatic lung adenocarcinoma cell line.

Brain metastasis frequently occurs in cancer patients and is associated with a poor prognosis. We previously reported that S100B was highly expressed in PC14/B, a specific brain metastatic lung adenocarcinoma cell line, which suggests that it is associated with brain metastasis of lung cancer. However, the role of S100B in brain metastasis remains to be elucidated. In this study, using PC14/B cell line, we found that siRNA mediated depletion of S100B in PC14/B cells led to notable differences in cell proliferation, apoptosis, cell cycle progression, colony formation ability, cell migratory and invasive activity compared with the mock-transfected cells. Therefore, our data suggest that S100B promotes the brain metastasis of lung adenocarcinoma by promoting cell proliferation, preventing apoptosis and increasing cell migration and invasion.

The effect of down regulation of calcineurin Aα by lentiviral vector-mediated RNAi on the biological behavior of small-cell lung cancer and its bone metastasis.

Bone is the third most common site of cancer metastasis. Over 30 to 40% of lung cancers can develop skeletal metastasis and no effective curative therapy exists in clinic cases. Previously we screened the different expression of proteins between SBC-5 cells and SBC-3 cells by proteomic study methods (MALDI-TOF/TOF-MS) and found that calcineurin (hereafter referred as Cn) overexpresses in SBC-5 which has special priority in metastasis to bone in a multiple-organ metastasis mice model. However the roles of Cn in osteotropism of SCLC remain to be elucidated. At present study, we decrease CnAα expression in SBC-5 by lentiviral vector-mediated RNAi and found that down regulation of CnAα gene expression can decrease the proliferation and colony formation rate, impede the cell cycle progression, reduce the cell migration and invasion, and inhibit cells adhering to bone matrix, but not change the apoptosis rate of SBC-5 in vitro. In vivo down or up regulation of CnAα gene expression can only decrease or increase the bone metastasis rate, but not affect the metastasis rate to the visceral organs. Our research reveals that CnAα is closely related to the osteotropism metastasis of SCLC and a candidate tumor promotor gene for developing bone metastases.

Calcineurin promotes proliferation, migration, and invasion of small cell lung cancer.

A novel role for calcineurin (Cn) has been reported recently regarding the oncogenic potential in pancreatic and colorectal cancer. The aim of this study was to investigate the putative causal role calcineurin could play in the development of lung cancer with bone metastases. We found that CnAalpha, an isoform of calcineurin, was significantly overexpressed in lung cancer tissues with bone metastasis as compared to tumors with non-bone metastases as investigated by RT-PCR. Strong nuclear staining of tumor cells was observed in small cell lung cancer tissues with bone metastasis. Conversely, cytoplasmic staining of tumor cells was observed in small cell lung cancer tissues with non-bone metastasis. Western blots of nuclear proteins from lung cancer tissues indicated that CnAalpha was highly expressed in lung cancer tissues with bone metastases, but not in those with non-bone metastases. In vitro, it was demonstrated that the CnAalpha gene obviously promoted cell proliferation and inhibited cell apotosis. The CnAalpha gene affected the cell cycle and promoted G1[Symbol: see text]S transition in SBC-3 cells. Transfection with the CnAalpha gene promoted cell migration and invasion. These results indicated that CnAalpha may affect the biological behavior of the human small cell lung cancer cell line SBC-3 in vitro and may be a candidate tumor promotor gene for developing bone metastases.