Recent progress in the development of hypoxia-inducible factor 2α (HIF-2α) modulators: Inhibitors, agonists, and degraders (2009-2024).

Hypoxia-inducible factor 2α (HIF-2α) is a critical transcription factor that regulates cellular responses under hypoxic conditions. In situations of insufficient oxygen supply or patients with Von Hippel-Lindau (VHL) mutations, HIF-2α accumulates and forms a heterodimeric complex with aryl hydrocarbon receptor nuclear translocator (ARNT, or HIF-ß). This complex further binds to coactivator p300 and interacts with hypoxia response elements (HREs) on the DNA of downstream target genes, regulating the transcription of a variety of genes (e.g. VEGFA, CCND1, CXCR4, SLC2A1, etc) involved in various processes like angiogenesis, mitochondrial metabolism, cell proliferation, and metastasis. Targeting HIF-2α holds great promise for effectively addressing solid tumors associated with aberrant oxygen-sensing pathways and hypoxia mechanisms, offering broad application prospects. In this review, we provide an overview of recent advancements (2009-2024) in HIF-2α modulators such as inhibitors, agonists, and degraders for cancer therapy. Additionally, we discuss in detail the challenges and future directions regarding HIF-2α modulators.

Discovery of Small and Bifunctional Molecules Targeting PD-L1/CD73 for Cancer Dual Immunotherapy.

In this work, a series of bifunctional PD-L1/CD73 (cluster of differentiation 73) small-molecule inhibitors were designed and synthesized. Among them, CC-5 showed the strongest PD-L1 inhibitory effects with an IC50 of 6 nM and potent anti-CD73 activity with an IC50 of 0.773 µM. The high PD-L1/CD73 inhibitory activity of CC-5 was further confirmed by SPR assays with KD of 182 nM for human PD-L1 and 101 nM for CD73, respectively. Importantly, CC-5 significantly suppressed tumor growth in a CT26 and B16-F10 tumor model with TGI of 64.3% and 39.6%, respectively. Immunohistochemical (IHC) and flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) indicated that CC-5 exerted anticancer effects via activating the tumor immune microenvironment. Collectively, CC-5 represents the first dual PD-L1/CD73 inhibitor worthy of further research as a bifunctional immunotherapeutic agent.

Recent Progress in DNA Damage Response-Targeting PROTAC Degraders.

DNA damage response (DDR) defects in cells play a crucial role in tumor development by promoting DNA mutations. These mutations create vulnerabilities specific to cancer cells, which can be effectively targeted through synthetic lethality-based therapies. To date, numerous small molecule DDR inhibitors have been identified, and some of them have already been approved for clinical use. However, due to the complexity of the tumor microenvironment, mutations may occur in the amino acid residues of DDR targets. These mutations can affect the efficacy of small molecule inhibitors targeting DDR pathways. Therefore, researchers have turned their attention to next-generation DNA damage repair modulators, particularly those based on PROTAC technology. From this perspective, we overviewed the recent progress on DDR-targeting PROTAC degraders for cancer therapy. In addition, we also summarized the biological functions of different DDR targets. Finally, the challenges and future directions for DDR-target PROTAC degraders are also discussed in detail.

Autophagy plays a pro-apoptotic role in arsenic trioxide-induced cell death of liver cancer.

OBJECTIVE: The effects of arsenic trioxide (As2O3) on hepatocellular carcinoma have been documented widely. Autophagy plays dual roles in the survival and death of cancer cells. Therefore, we investigated the exact role of autophagy in As2O3-induced apoptosis in liver cancer cells. METHODS: The viability of hepatoma cells was determined using the MTT assay with or without fetal bovine serum. The rate of apoptosis in liver cancer cells treated with As2O3 was evaluated using flow cytometry, Hoechst 33258 staining, and TUNEL assays. The rate of autophagy among liver cancer cells treated with As2O3 was detected using immunofluorescence, Western blot assay and transmission electron microscopy. RESULTS: Upon treatment with As2O3, the viability of HepG2 and SMMC-7721 cells was decreased in a time- and dose-dependent manner. The apoptosis rates of both liver cancer cell lines increased with the concentration of As2O3, as shown by flow cytometry. Apoptosis in liver cancer cells treated with As2O3 was also shown by the activation of the caspase cascade and the regulation of Bcl-2/Bax expression. Furthermore, As2O3 treatment induced autophagy in liver cancer cells; this finding was supported by Western blot, immunofluorescence of LC3-II and beclin 1, and transmission electron microscopy. In liver cancer cells, As2O3 inhibited the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signal pathway that plays a vital role in both apoptosis and autophagy. The PI3K activator SC-79 partially reversed As2O3-induced autophagy and apoptosis. Furthermore, inhibiting autophagy with 3-methyladenine partially reversed the negative effects of As2O3 on cell viability. Serum starvation increased autophagy and amplified the effect of As2O3 on cell death. CONCLUSION: As2O3 induces apoptosis and autophagy in liver cancer cells. Autophagy induced by As2O3 may have a proapoptotic effect that helps to reduce the viability of liver cancer cells. This study provides novel insights into the effects of As2O3 against liver cancer. Please cite this article as: Deng ZT, Liang SF, Huang GK, Wang YQ, Tu XY, Zhang YN, Li S, Liu T, Cheng BB. Autophagy plays a pro-apoptotic role in arsenic trioxide-induced cell death of liver cancer. J Integr Med. 2024; 22(3): 295-302.

Discovery of Novel Heterotricyclic Compounds as DNA-Dependent Protein Kinase (DNA-PK) Inhibitors with Enhanced Chemosensitivity, Oral Bioavailability, and the Ability to Potentiate Cancer Immunotherapy.

In this work, a novel series of heterotricyclic DNA-PK inhibitors were rationally designed, synthesized, and assessed for their biological activity. In the DNA-PK biochemical assay, most compounds displayed potent enzymatic activity, with IC50 values between 0.11 and 71.5 nM. Among them, SK10 exhibited the most potent DNA-PK-inhibitory activity (IC50 = 0.11 nM). Studies of the mechanism of action indicated that SK10 could lower γH2A.X expression levels and demonstrate optimal synergistic antiproliferative activity against Jurkat cells (IC50 = 25 nM) when combined with doxorubicin. Importantly, in CT26 and B16-F10 tumor-bearing mouse models, the combination therapies of SK10 with chemotherapeutic drug doxorubicin, a PD-L1 antibody, and SWS1 (a potent PD-L1 small-molecule inhibitor) demonstrated superior synergistic anticancer and potential immunomodulatory effects. Furthermore, SK10 possessed favorable in vivo pharmacokinetic properties [e.g., oral bioavailability (F) = 31.8%]. Taken together, SK10 represents a novel heterotricyclic DNA-PK inhibitor with antitumor immune effects and favorable pharmacokinetics.

A Hypoxia-Related miRNA-mRNA Signature for Predicting the Response and Prognosis of Transcatheter Arterial Chemoembolization in Hepatocellular Carcinoma.

Purpose: Transcatheter arterial chemoembolization (TACE) is commonly used in the treatment of hepatocellular carcinoma (HCC). However, not all patients respond to this treatment. TACE typically leads to hypoxia in the tumor microenvironment. Therefore, we aimed to construct a prognostic model based on hypoxia-related differentially expressed microRNA (miRNAs) in hepatocellular carcinoma (HCC) and to investigate the potential target mRNAs for predicting TACE response. Methods: The hypoxia-related miRNAs (HRMs) were identified in liver cancer cells, then global test was performed to further select the miRNAs which were associated with recurrence and vascular invasion. A prognostic model was constructed based on multivariate Cox regression analysis; qRT-PCR analysis was used to validate the differentially expressed miRNAs in HCC cell lines under hypoxic condition. We further identified the putative target genes of the miRNAs and investigate the relationship between the target genes and TACE response, immune cells infiltration. Results: We established a HRMs prognostic model for HCC patients, containing two miRNAs (miR-638, miR-501-5p), the patients with high-HRMs score showed worse survival in discovery and validation cohort; qRT-PCR analysis confirmed that these two miRNAs are up-regulated in hepatoma cells under hypoxic condition. Furthermore, four putative target genes of these two miRNAs were identified (ADH1B, CTH, FTCD, RCL1), which were significantly associated with TACE response, immune score, immunosuppressive immune cells infiltration, PDCD1 and CTLA4. Conclusion: The HCC-HRMs signature may be utilized as a promising prognostic factor and may have implications for guiding TACE and immune therapy.

Alternative mRNA splicing events and regulators in epidermal differentiation.

Alternative splicing (AS) of messenger RNAs occurs in ∼95% of multi-exon human genes and generates diverse RNA and protein isoforms. We investigated AS events associated with human epidermal differentiation, a process crucial for skin function. We identified 6,413 AS events, primarily involving cassette exons. We also predicted 34 RNA-binding proteins (RBPs) regulating epidermal AS, including 19 previously undescribed candidate regulators. From these results, we identified FUS as an RBP that regulates the balance between keratinocyte proliferation and differentiation. Additionally, we characterized the function of a cassette exon AS event in MAP3K7, which encodes a kinase involved in cell signaling. We found that a switch from the short to long isoform of MAP3K7, triggered during differentiation, enforces the demarcation between proliferating basal progenitors and overlying differentiated strata. Our findings indicate that AS occurs extensively in the human epidermis and has critical roles in skin homeostasis.

Oleanolic acid inhibits hypoxic tumor-derived exosomes-induced premetastatic niche formation in hepatocellular carcinoma by targeting ERK1/2-NFκB signaling.

BACKGROUND: Pulmonary premetastatic niche (PMN) formation plays a key role in the lung metastasis of hepatocellular carcinoma (HCC). Hypoxia promotes the secretion of tumor-derived exosomes (TDEs) and facilitates the formation of PMN. However, the mechanisms remain unexplored. METHODS: TDEs from normoxic (N-TDEs) or hypoxic (H-TDEs) HCC cells were used to induce fibroblast activation in vitro and PMN formation in vivo. Oleanolic acid (OA) was intragastrically administered to TDEs-preconditioned mice. Bioinformatics analysis and drug affinity responsive target stability (DARTS) assays were performed to identify targets of OA in fibroblasts. RESULTS: H-TDEs induced activation of pulmonary fibroblasts, promoted formation of pulmonary PMN and subsequently facilitated lung metastasis of HCC. OA inhibited TDEs-induced PMN formation and lung metastasis and suppressed TDEs-mediated fibroblast activation. MAPK1 and MAPK3 (ERK1/2) were the potential targets of OA. Furthermore, H-TDEs enhanced ERK1/2 phosphorylation in fibroblasts in vitro and in vivo, which was suppressed by OA treatment. Blocking ERK1/2 signaling with its inhibitor abated H-TDEs-induced activation of fibroblasts and PMN formation. H-TDEs-induced phosphorylation of ERK1/2 in fibroblasts touched off the activation NF-κB p65, which was mitigated by OA. In addition, the ERK activator C16-PAF recovered the activation of ERK1/2 and NF-κB p65 in H-TDEs-stimulated MRC5 cells upon OA treatment. CONCLUSION: The present study offers insights into the prevention of TDEs-induced PMN, which has been insufficiently investigated. OA suppresses the activation of inflammatory fibroblasts and the development of pulmonary PMN by targeting ERK1/2 and thereby has therapeutic potential in the prevention of lung metastasis of HCC.

HDAC-targeting epigenetic modulators for cancer immunotherapy.

HDAC inhibitors, which can inhibit the activity of HDAC enzymes, have been extensively studied in tumor immunotherapy and have shown potential therapeutic effects in cancer immunotherapy. To date, numerous small molecule HDAC inhibitors have been identified, but many of them suffer from limited clinical efficacy and serious toxicity. Hence, HDAC inhibitor-based combination therapies, and other HDAC modulators (e.g. PROTAC degraders, dual-acting agents) have attracted great attention with significant advancements achieved in the past few years due to their superior efficacy compared to single-target HDAC inhibitors. In this review, we overviewed the recent progress on HDAC-based drug discovery with a focus on HDAC inhibitor-based drug combination therapy and other HDAC-targeting strategies (e.g. selective HDAC inhibitors, HDAC-based dual-target inhibitors, and PROTAC HDAC degraders) for cancer immunotherapy. In addition, we also summarized the reported co-crystal structures of HDAC inhibitors in complex with their target proteins and the binding interactions. Finally, the challenges and future directions for HDAC-based drug discovery in cancer immunotherapy are also discussed in detail.

Clinical characteristics of 14 cases of Chlamydia psittaci pneumonia.

Among 14 patients with C. psittaci pneumonia, there were 9 critical and 5 non-critical cases. Ten patients improved clinically and were discharged to home; however, four patients died. Seven patients had a history of contact with birds or poultry. All 14 patients had a high fever as the presenting symptom, but most had a normal white blood cell count. Most of the patients had a significant increase in high-sensitivity C-reactive protein and procalcitonin levels. The lymphocyte count in the critical group was considerably lower than in the non-critical group. Patients in the critical group were more advanced in age than in the non-critical group. In addition, serum urea nitrogen, creatinine, procalcitonin, and lactate dehydrogenase levels were significantly higher in the critical group than in the non-critical group (P<0.05). The 4 patients who died had significantly increased procalcitonin levels compared to the 10 patients who survived (P<0.05). In summary, a high fever is usually the presenting complaint of patients with C. psittaci pneumonia. Such patients often progress to severe disease; however, early diagnostic confirmation by mNGS and appropriate treatment dramatically improve the prognosis. Age, lymphocyte count, procalcitonin, blood urea nitrogen, creatinine, and lactate dehydrogenase levels were shown to predict disease severity.

The role of dysregulated mRNA translation machinery in cancer pathogenesis and therapeutic value of ribosome-inactivating proteins.

Dysregulated protein synthesis is a hallmark of tumors. mRNA translation reprogramming contributes to tumorigenesis, which is fueled by abnormalities in ribosome formation, tRNA abundance and modification, and translation factors. Not only malignant cells but also stromal cells within tumor microenvironment can undergo transformation toward tumorigenic phenotypes during translational reprogramming. Ribosome-inactivating proteins (RIPs) have garnered interests for their ability to selectively inhibit protein synthesis and suppress tumor growth. This review summarizes the role of dysregulated translation machinery in tumor development and explores the potential of RIPs in cancer treatment.

Hypoxia-induced exosomes facilitate lung pre-metastatic niche formation in hepatocellular carcinoma through the miR-4508-RFX1-IL17A-p38 MAPK-NF-κB pathway.

Background: Hypoxia plays an important role in the lung metastasis of hepatocellular carcinoma (HCC). However, the process by which hypoxia promotes the formation of a pre-metastatic niche (PMN) and its underlying mechanism remain unclear. Methods: Exosomes derived from normoxic and hypoxic HCC cells were collected to induce fibroblast activation in vitro and PMN formation in vivo. The micro RNA (miR) profiles of the exosomes were sequenced to identify differentially expressed miRNAs. Gain- and loss-of-function analyses were performed to investigate miR-4508 function. Dual-luciferase, western blotting, and real-time reverse transcription-PCR analyses were used to identify the direct targets of miR-4508 and its downstream signaling pathways. To demonstrate the roles of hypoxic tumor-derived exosomes (H-TDEs) and miR-4508 in the lung metastasis of liver cancer, H22 tumor cells were injected through the tail vein of mice. Blood plasma-derived exosomes from patients with HCC who underwent transarterial chemoembolization (TACE) were applied to determine clinical correlations. Results: We demonstrated that H-TDEs activated lung fibroblasts and facilitated PMN formation, thereby promoting lung metastasis in mice. Screening for upregulated exosomal miRNAs revealed that miR-4508 and its target, regulatory factor X1 (RFX1), were involved in H-TDE-induced lung PMN formation. Moreover, miR-4508 was significantly upregulated in plasma exosomes derived from patients with HCC after TACE. We confirmed that the p38 MAPK-NF-κB signaling pathway is involved in RFX1 knockdown-induced fibroblast activation and PMN formation. In addition, IL17A, a downstream target of RFX1, was identified as a link between RFX1 knockdown and p38 MAPK activation in fibroblasts. Conclusion: Hypoxia enhances the release of TDEs enriched with miR-4508, thereby promoting lung PMN formation by targeting the RFX1-IL17A-p38 MAPK-NF-κB pathway. These findings highlight a novel mechanism underlying hypoxia-induced pulmonary metastasis of HCC.

Discovery of Novel d-(+)-Biotin-Conjugated Resorcinol Dibenzyl Ether-Based PD-L1 Inhibitors for Targeted Cancer Immunotherapy.

In this work, we rationally designed, synthesized, and evaluated a series of novel d-(+)-biotin-conjugated PD-L1 inhibitors for targeted cancer therapy. Among them, SWS1 exhibited the highest anti-PD-1/PD-L1 activity with an IC50 of 1.8 nM. In addition, SWS1 dose-dependently promoted tumor cell death in a HepG2/Jurkat cell co-culture model. Importantly, SWS1 displayed high antitumor efficacy in a B16-F10 mouse model with tumor growth inhibition of 66.1%, which was better than that of P18 (44.3%). Furthermore, SWS1 exerted antitumor effects by increasing the number of tumor-infiltrating lymphocytes and reducing the expression of PD-L1 in tumor tissues. Moreover, tissue distribution studies revealed a substantial accumulation of SWS1 in tumors (404.1 ng/mL). Lastly, the safety profiles of SWS1 were better (e.g., less immune-mediated colitis) than those of P18, indicating the advantages of biotin-enabled tumor targeting capability. Taken together, our results suggest that these novel tumor-targeted PD-L1 inhibitors are worthy of further investigation as potential anticancer agents for targeted cancer immunotherapy.

Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/ß-catenin pathway under hypoxia.

OBJECTIVE: Jiedu Recipe (JR), a Chinese herbal remedy, has been shown to prolong overall survival time and decrease recurrence and metastasis rates in patients with hepatocellular carcinoma (HCC). This work investigated the mechanism of JR in HCC treatment. METHODS: The chemical constituents of JR were detected using liquid chromatography-mass spectrometry. The potential anti-HCC mechanism of JR was screened using network pharmacology and messenger ribonucleic acid (mRNA) microarray chip assay, followed by experimental validation in human HCC cells (SMMC-7721 and Huh7) in vitro and a nude mouse subcutaneous transplantation model of HCC in vivo. HCC cell characteristics of proliferation, migration and invasion under hypoxic setting were investigated using thiazolyl blue tetrazolium bromide, wound healing and Transwell assays, respectively. Image-iT™ Hypoxia Reagent was added to reveal hypoxic conditions. Stem cell sphere formation assay was used to detect the stemness. Epithelial-mesenchymal transition (EMT) markers like E-cadherin, vimentin and α-smooth muscle actin, and pluripotent transcription factors including nanog homeobox, octamer-binding transcription factor 4, and sex-determining region Y box protein 2 were analyzed using Western blotting and real-time polymerase chain reaction. Western blot was performed to ascertain the anti-HCC effect of JR under hypoxia involving the Wnt/ß-catenin pathway. RESULTS: According to network pharmacology and mRNA microarray chip analysis, JR may potentially act on hypoxia and inhibit the Wnt/ß-catenin pathway. In vitro and in vivo experiments showed that JR significantly decreased hypoxia, and suppressed HCC cell features of proliferation, migration and invasion; furthermore, the hypoxia-induced increases in EMT and stemness marker expression in HCC cells were inhibited by JR. Results based on the co-administration of JR and an agonist (LiCl) or inhibitor (IWR-1-endo) verified that JR suppressed HCC cancer stem-like properties under hypoxia by blocking the Wnt/ß-catenin pathway. CONCLUSION: JR exerts potent anti-HCC effects by inhibiting cancer stemness via abating the Wnt/ß-catenin pathway under hypoxic conditions. Please cite this article as: Guo BJ, Ruan Y, Wang YJ, Xiao CL, Zhong ZP, Cheng BB, Du J, Li B, Gu W, Yin ZF. Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/ß-catenin pathway under hypoxia. J Integr Med. 2023; 21(5): 474-486.

Targeting tumor-derived exosome-mediated premetastatic niche formation: The metastasis-preventive value of traditional Chinese medicine.

Tumor-derived exosome (TDE)-mediated premetastatic niche (PMN) formation is a potential mechanism underlying the organotropic metastasis of primary tumors. Traditional Chinese medicine (TCM) has shown considerable success in preventing and treating tumor metastasis. However, the underlying mechanisms remain elusive. In this review, we discussed PMN formation from the perspectives of TDE biogenesis, cargo sorting, and TDE recipient cell alterations, which are critical for metastatic outgrowth. We also reviewed the metastasis-preventive effects of TCM, which act by targeting the physicochemical materials and functional mediators of TDE biogenesis, regulating the cargo sorting machinery and secretory molecules in TDEs, and targeting the TDE-recipient cells involved in PMN formation.

Role of exosomes in hepatocellular carcinoma and the regulation of traditional Chinese medicine.

Hepatocellular carcinoma (HCC) usually occurs on the basis of chronic liver inflammatory diseases and cirrhosis. The liver microenvironment plays a vital role in the tumor initiation and progression. Exosomes, which are nanometer-sized membrane vesicles are secreted by a number of cell types. Exosomes carry multiple proteins, DNAs and various forms of RNA, and are mediators of cell-cell communication and regulate the tumor microenvironment. In the recent decade, many studies have demonstrated that exosomes are involved in the communication between HCC cells and the stromal cells, including endothelial cells, macrophages, hepatic stellate cells and the immune cells, and serve as a regulator in the tumor proliferation and metastasis, immune evasion and immunotherapy. In addition, exosomes can also be used for the diagnosis and treatment HCC. They can potentially serve as specific biomarkers for early diagnosis and drug delivery vehicles of HCC. Chinese herbal medicine, which is widely used in the prevention and treatment of HCC in China, may regulate the release of exosomes and exosomes-mediated intercellular communication. In this review, we summarized the latest progresses on the role of the exosomes in the initiation, progression and treatment of HCC and the potential value of Traditional Chinese medicine in exosomes-mediated biological behaviors of HCC.

Corrigendum to "The Chinese Medicine, Jiedu Recipe, Inhibits the Epithelial Mesenchymal Transition of Hepatocellular Carcinoma via the Regulation of Smad2/3 Dependent and Independent Pathways".

[This corrects the article DOI: 10.1155/2018/5629304.].

Discovery of novel 7,8-dihydropteridine-6(5H)-one-based DNA-PK inhibitors as potential anticancer agents via scaffold hopping strategy.

DNA-dependent protein kinase (DNA-PK) is an essential element in the DNA damage response (DDR) pathway and has been regarded as a druggable target for antineoplastic agents. Starting from AZD-7648, a potent DNA-PK inhibitor being investigated in phase II clinical trials for advanced cancer treatment, two series of DNA-PK inhibitors were rationally designed via scaffold hopping strategy, synthesized, and assessed for their biological activity. Most compounds exhibited potent biochemical activity on DNA-PK enzymatic assay with IC50 values below 300 nM. Among these compounds, DK1 showed the best DNA-PK-inhibitory potency (IC50 = 0.8 nM), slightly better than that of AZD-7648 (IC50 = 1.58 nM). Mode of action studies revealed that compound DK1 decreased the expression levels of γH2A.X and demonstrated synergistic antiproliferative activity against a series of cancer cell lines when used in combination with doxorubicin. Moreover, DK1 showed reasonable in vitro drug-like properties and favorable in vivo pharmacokinetics as an oral drug candidate. Importantly, the combination therapy of DK1 with DNA double-strand break (DSB)-inducing agent doxorubicin showed synergistic anticancer efficacy in the HL-60 xenograft model with a tumor growth inhibition (TGI) of 52.4% and 62.4% for tumor weight and tumor volume, respectively. In conclusion, DK1 is a novel DNA-PK inhibitor with great promise for further study.