Kiyohiro Ando

Cancer cell plasticity is tightly linked to therapeutic resistance in urothelial carcinoma (UC). Signal transducer and activator of transcription 3 (STAT3) and CD44 play crucial roles in plasticity, but their potential crosstalk in the therapeutic context has not yet been fully elucidated. This study aimed to unveil the shared and distinct roles of STAT3 and CD44 and obtain a better understanding of targeted therapy in UC. T24 bladder cancer cells were genetically ablated for STAT3 and CD44 and their plasticity was subsequently assessed. Phosphorylated-STAT3 (pSTAT3) and CD44 expression was determined using tissue microarrays in 16 patients who received pembrolizumab therapy for UC, and the association of their expression levels with patient prognosis was investigated. Ablation of STAT3 or CD44 expression resulted in the dysregulation of vimentin expression and promotion of cell spheroid viability. Patients with imbalanced pSTAT3 and CD44 expression had significantly shorter progression-free survival and overall survival. Functional imbalance of STAT3 and CD44 may promote cancer cell plasticity and result in impaired pembrolizumab response in patients with advanced UC.

Background/Aim: Pembrolizumab, a second-line therapy for platinum-refractory advanced urothelial carcinoma (UC), is needed to improve objective response rate. Hence, it is crucial to identify optimal predictive biomarkers of responses. This study aimed to clarify the predictive value and role of signal transducer and activator of transcription 3 (STAT3) in selecting patients with advanced UC who might benefit clinically from pembrolizumab therapy. Patients and Methods: We retrospectively analyzed 31 patients who received pembrolizumab therapy for UC. STAT3, phosphorylated STAT3 (p-STAT3), and PD-L1 expression were determined using tissue microarrays constructed from patient-derived specimens, and the association of these expression levels with overall survival was analyzed. We assessed the functional role of STAT3 in bladder cancer cell lines in response to interferon-gamma (IFN-γ). Results: Patients with high STAT3 or p-STAT3 expression, and high platelet-to-lymphocyte ratio (PLR) (n=6) had a significantly shorter OS; in the other patients (n=25), high STAT3 or p-STAT3 expression was significantly associated with improved prognosis. IFN-γ-induced apoptosis was partially dependent on STAT3 in T24 cells but not in JMSU1 cells. Conclusion: In patients with advanced UC, STAT3 plays a key role in mediating the efficacy of pembrolizumab through apoptosis in response to IFN-γ.

Identifying the vulnerability of altered DNA repair machinery that displays synthetic lethality with MYCN amplification is a therapeutic rationale in unfavourable neuroblastoma. However, none of the inhibitors for DNA repair proteins are established as standard therapy in neuroblastoma. Here, we investigated whether DNA-PK inhibitor (DNA-PKi) could inhibit the proliferation of spheroids derived from neuroblastomas of MYCN transgenic mice and MYCN-amplified neuroblastoma cell lines. DNA-PKi exhibited an inhibitory effect on the proliferation of MYCN-driven neuroblastoma spheroids, whereas variable sensitivity was observed in those cell lines. Among them, the accelerated proliferation of IMR32 cells was dependent on DNA ligase 4 (LIG4), which comprises the canonical non-homologous end-joining pathway of DNA repair. Notably, LIG4 was identified as one of the worst prognostic factors in patients with MYCN-amplified neuroblastomas. It may play complementary roles in DNA-PK deficiency, suggesting the therapeutic potential of LIG4 inhibition in combination with DNA-PKi for MYCN-amplified neuroblastomas to overcome resistance to multimodal therapy.

Background/Aim: Several prognostic risk factors have been recognized when using cisplatin-based conventional chemotherapy for the treatment of advanced urothelial carcinoma (UC); these include performance status (PS), liver metastasis, hemoglobin (Hb) levels, time from prior chemotherapy (TFPC), and other systemic inflammation scores including neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR). However, the benefit of these indicators for predicting outcome of immune checkpoint inhibitors is not fully understood. Here, we investigated the predictive value of the indicators in patients who received pembrolizumab for the treatment of advanced UC. Patients and Methods: Seventy-five patients who received pembrolizumab treatment for advanced UC were included. The Karnofsky PS, liver metastasis, hemoglobin levels, TFPC, NLR, and PLR were analyzed, and their relationship with overall survival (OS) was determined. Results: All factors were highlighted as significant prognostic indicators for OS in the univariate proportional regression analysis (p<0.05 for each). Multivariate analysis revealed that Karnofsky PS and liver metastasis were independent prognostic indicators for OS (p<0.01) but were applicable only for a small number of patients. Notably, the combined analysis with low Hb levels and high PLR was significantly associated with OS in patients who could gain less benefit from pembrolizumab at a median of 6.6 [95% confidence interval (CI)=4.2-9.0] versus 15.1 (95% CI=12.4-17.8) months (p=0.002). Conclusion: The combination of Hb levels and PLR may be a broadly applicable indicator for the outcome of pembrolizumab as second-line chemotherapy in patients with advanced UC.

The Runt-related transcription factor (RUNX) family, which is essential for the differentiation of cells of neural crest origin, also plays a potential role in neuroblastoma tumorigenesis. Consecutive studies in various tumor types have demonstrated that the RUNX family can play either pro-tumorigenic or anti-tumorigenic roles in a context-dependent manner, including in response to chemotherapeutic agents. However, in primary neuroblastomas, RUNX3 acts as a tumor-suppressor, whereas RUNX1 bifunctionally regulates cell proliferation according to the characterized genetic and epigenetic backgrounds, including MYCN oncogenesis. In this review, we first highlight the current knowledge regarding the mechanism through which the RUNX family regulates the neurotrophin receptors known as the tropomyosin-related kinase (Trk) family, which are significantly associated with neuroblastoma aggressiveness. We then focus on the possible involvement of the RUNX family in functional alterations of the p53 family members that execute either tumor-suppressive or dominant-negative functions in neuroblastoma tumorigenesis. By examining the tripartite relationship between the RUNX, Trk, and p53 families, in addition to the oncogene MYCN, we endeavor to elucidate the possible contribution of the RUNX family to neuroblastoma tumorigenesis for a better understanding of potential future molecular-based therapies.

In the present study, we found that EZH1 depletion in MYCN‐amplified neuroblastoma cells resulted in significant cell death as well as xenograft inhibition. EZH1 depletion decreased the level of H3K27me1; the interaction and protein stabilization of MYCN and EZH1 appear to play roles in epigenetic transcriptional regulation. Transcriptome analysis of EZH1‐depleted cells resulted in downregulation of the cell cycle progression‐related pathway. In particular, Gene Set Enrichment Analysis revealed downregulation of reactome E2F‐mediated regulation of DNA replication along with key genes of this process, TYMS, POLA2, and CCNA1. TYMS and POLA2 were transcriptionally activated by MYCN and EZH1‐related epigenetic modification. Treatment with the EZH1/2 inhibitor UNC1999 also induced cell death, decreased H3K27 methylation, and reduced the levels of TYMS in neuroblastoma cells. Previous reports indicated neuroblastoma cells are resistant to 5‐fluorouracil (5‐FU) and TYMS (encoding thymidylate synthetase) has been considered the primary site of action for folate analogues. Intriguingly, UNC1999 treatment significantly sensitized MYCN‐amplified neuroblastoma cells to 5‐FU treatment, suggesting that EZH inhibition could be an effective strategy for development of a new epigenetic treatment for neuroblastoma.

Editorial: Neuroblastoma: Signal Crosstalk and Future Insight Into Clinical Trials

Background/Aim: This study aimed to determine useful predictive factors for selecting patients with advanced urothelial carcinoma (UC) who might benefit clinically from treatment with pembrolizumab. Patients and Methods: We retrospectively analyzed 54 patients who underwent pembrolizumab treatment for UC. The hemoglobin, albumin, lymphocyte and platelet (HALP) score, neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) were calculated as indices of systemic inflammatory response, and the relationships between these scores and the initial tumor response or overall survival, as well as other clinicopathological factors, were assessed. Results: High NLR and PLR were associated with a poor initial tumor response to pembrolizumab. A HALP score <30.05 and a PLR ≥173.73 were associated with worse overall survival. In the multivariate Cox regression analysis, a high PLR was a significant independent prognostic factor for unfavorable outcomes. Conclusion: A high pretreatment PLR may be a valuable indicator for choosing therapy other than pembrolizumab in patients with advanced UC.

Checkpoint kinase 1 (CHK1) plays a key role in genome surveillance and integrity throughout the cell cycle. Selective inhibitors of CHK1 (CHK1i) are undergoing clinical evaluation for various human malignancies, including neuroblastoma. In this study, one CHK1i‐sensitive neuroblastoma cell line, CHP134, was investigated, which characteristically carries MYCN amplification and a chromosome deletion within the 10q region. Among several cancer‐related genes in the chromosome 10q region, mRNA expression of fibroblast growth factor receptor 2 (FGFR2) was altered in CHP134 cells and associated with an unfavorable prognosis of patients with neuroblastoma. Induced expression of FGFR2 in CHP134 cells reactivated downstream MEK/ERK signaling and resulted in cells resistant to CHK1i‐mediated cell growth inhibition. Consistently, the MEK1/2 inhibitor, trametinib, potentiated CHK1 inhibitor–mediated cell death in these cells. These results suggested that FGFR2 loss might be prone to highly effective CHK1i treatment. In conclusion, extreme cellular dependency of ERK activation may imply a possible application for the MEK1/2 inhibitor, either as a single inhibitor or in combination with CHK1i in MYCN‐amplified neuroblastomas.