Dead Cells Don’t Form Tumors: HIF-Dependent Cytotoxins

Elimination or reduction of tumor burden is the primary goal of cancer therapy. Strategies to achieve this goal with the fewest adverse effects to the patient are an area of intense investigation. Elevated protein levels of hypoxia-inducible factor (HIF) are commonly found in solid tumors, while rarely found in healthy tissue. Numerous studies have suggested that HIF activity is essential for the development of solid tumors. Thus, inhibition of HIF represents an attractive therapeutic target for eradicating tumors. The search for small molecules that target and inhibit HIF activity is currently underway. We propose an alternate approach: to directly target and kill HIF-activated tumor cells. This approach is advantageous in that cells with activated HIF will be eliminated directly. Specific elimination of HIF-activated cells represents a potential mechanism for inhibiting tumor growth, with the potential advantage of sparing the patient of the normal tissue toxicity associated with current treatment options.     

Assessment of hypoxia inducible factor levels in cancer cell lines upon hypoxic induction using a novel reporter construct

Hypoxia Inducible Factor (HIF) signaling pathway is important for tumor cells with limited oxygen supplies, as it is shown to be involved in the process of proliferation and angiogenesis. Given its pivotal role in cancer biology, robust assays for tracking changes in HIF expression are necessary for understanding its regulation in cancer as well as developing therapies that target HIF signaling. Here we report a novel HIF reporter construct containing tandem repeats of minimum HIF binding sites upstream of eYFP coding sequence. We show that the reporter construct has an excellent signal to background ratio and the reporter activity is HIF dependent and directly correlates with HIF protein levels. By utilizing this new construct, we assayed HIF activity levels in different cancer cell lines cultured in various degrees of hypoxia. This analysis reveals a surprising cancer cell line specific variation of HIF activity in the same level of hypoxia. We further show that in two cervical cancer cell lines, ME180 and HeLa, the different HIF activity levels observed correlate with the levels of hsp90, a cofactor that protects HIF against VHL-independent degradation. This novel HIF reporter construct serves as a tool to rapidly define HIF activity levels and therefore the therapeutic capacity of potential HIF repressors in individual cancers.     

A novel benzimidazole analogue inhibits the hypoxia-inducible factor (HIF)-1 pathway

Hypoxia-inducible factor (HIF)-1 is a therapeutic target in solid tumors. We report the novel benzimidazole analogue AC1-004, obtained from a chemical library using an HRE-dependent cell-based assay in colorectal carcinoma HCT-116 cells. The accumulation of hypoxia-induced HIF-1α was inhibited by compound AC1-004 in various cancer cells, including HCT-116, MDA-MB435, SK-HEP1, and Caki-1. Further, AC1-004 down-regulated VEGF and EPO, target genes of HIF-1, and inhibited in vitro tube formation of HUVEC, suggesting its potential inhibitory activity on angiogenesis. Importantly, AC1-004 was found to regulate the stability of HIF-1α through the Hsp90-Akt pathway, leading to the degradation of HIF-1α. An in vivo antitumor study demonstrated that AC1-004 reduced tumor size significantly (i.e., by 58.6%), without severe side effects. These results suggest the benzimidazole analogue AC1-004 is a novel HIF inhibitor that targets HIF-1α via the Hsp90-Akt pathway, and that it can be used as a new lead in developing anticancer drugs.     

LINE-1 methylation in plasma DNA as a biomarker of activity of DNA methylation inhibitors in patients with solid tumors

Multiple clinical trials are investigating the use of the DNA methylation inhibitors azacitidine and decitabine for the treatment of solid tumors. Clinical trials in hematological malignancies have shown that optimal activity does not occur at their maximum tolerated doses but selection of an optimal biological dose and schedule for use in solid tumor patients is hampered by the difficulty of obtaining tumor tissue to measure their activity. Here we investigate the feasibility of using plasma DNA to measure the demethylating activity of the DNA methylation inhibitors in patients with solid tumors. We compared four methods to measure LINE-1 and MAGE-A1 promoter methylation in T24 and HCT116 cancer cells treated with decitabine treatment and selected Pyrosequencing for its greater reproducibility and higher signal to noise ratio. We then obtained DNA from plasma, peripheral blood mononuclear cells, buccal mucosa cells and saliva from ten patients with metastatic solid tumors at two different time points, without any intervening treatment. DNA methylation measurements were not significantly different between time point 1 and time point 2 in patient samples. We conclude that measurement of LINE-1 methylation in DNA extracted from the plasma of patients with advanced solid tumors, using Pyrosequencing, is feasible and has low within patient variability. Ongoing studies will determine whether changes in LINE-1 methylation in plasma DNA occur as a result of treatment with DNA methylation inhibitors and parallel changes in tumor tissue DNA.     

NDUFA4L2 promotes glioblastoma progression,is associated with poor survival,and can be effectively targeted by apatinib

NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2 (NDUFA4L2) is a subunit of Complex I of the mitochondrial respiratory chain, which is important in metabolic reprogramming and oxidative stress in multiple cancers. However, the biological role and molecular regulation of NDUFA4L2 in glioblastoma (GBM) are poorly understood. Here, we found that NDUFA4L2 was significantly upregulated in GBM; the elevated levels were correlated with reduced patient survival. Gene knockdown of NDUFA4L2 inhibited tumor cell proliferation and enhanced apoptosis, while tumor cells initiated protective mitophagy in vitro and in vivo. We used lentivirus to reduce expression levels of NDUFA4L2 protein in GBM cells exposed to mitophagy blockers, which led to a significant enhancement of tumor cell apoptosis in vitro and inhibited the development of xenografted tumors in vivo. In contrast to other tumor types, NDUFA4L2 expression in GBM may not be directly regulated by hypoxia-inducible factor (HIF)-1α, because HIF-1α inhibitors failed to inhibit NDUFA4L2 in GBM. Apatinib was able to effectively target NDUFA4L2 in GBM, presenting an alternative to the use of lentiviruses, which currently cannot be used in humans. Taken together, our data suggest the use of NDUFA4L2 as a potential therapeutic target in GBM and demonstrate a practical treatment approach.Subject terms: CNS cancer, Mitophagy     

Human solid tumor xenografts in immunodeficient mice are vulnerable to lymphomagenesis associated with Epstein-Barr virus

Xenografting primary human solid tumor tissue into immunodeficient mice is a widely used tool in studies of human cancer biology; however, care must be taken to prove that the tumors obtained recapitulate parent tissue. We xenografted primary human hepatocellular carcinoma (HCC) tumor fragments or bulk tumor cell suspensions into immunodeficient mice. We unexpectedly observed that 11 of 21 xenografts generated from 16 independent patient samples resembled lymphoid neoplasms rather than HCC. Immunohistochemistry and flow cytometry analyses revealed that the lymphoid neoplasms were comprised of cells expressing human CD45 and CD19/20, consistent with human B lymphocytes. In situ hybridization was strongly positive for Epstein-Barr virus (EBV) encoded RNA. Genomic analysis revealed unique monoclonal or oligoclonal immunoglobulin heavy chain gene rearrangements in each B-cell neoplasm. These data demonstrate that the lymphoid neoplasms were EBV-associated human B-cell lymphomas. Analogous to EBV-associated lymphoproliferative disorders in immunocompromised humans, the human lymphomas in these HCC xenografts likely developed from reactivation of latent EBV in intratumoral passenger B lymphocytes following their xenotransplantation into immunodeficient recipient mice. Given the high prevalence of latent EBV infection in humans and the universal presence of B lymphocytes in solid tumors, this potentially confounding process represents an important pitfall of human solid tumor xenografting. This phenomenon can be recognized and avoided by routine phenotyping of primary tumors and xenografts with human leukocyte markers, and provides a compelling biological rationale for exclusion of these cells from human solid tumor xenotransplantation assays.     

Targeting Trop-2 in cancer: Recent research progress and clinical application

The development of new antitumor drugs depends mainly upon targeting tumor cells precisely. Trophoblast surface antigen 2 (Trop-2) is a type I transmembrane glycoprotein involved in Ca²⁺ signaling in tumor cells. It is highly expressed in various tumor tissues than in normal tissues and represents a novel and promising molecular target for caner targeted therapy. Up to now, the mechanisms and functions associated with Trop-2 have been extensively studied in a variety of solid tumors. According to these findings, Trop-2 plays an important role in cell proliferation, apoptosis, cell adhesion, epithelial-mesenchymal transition, as well as tumorigenesis and tumor progression. In addition, Trop-2 related drugs are also being developed widely. There are a number of Trop-2 related ADC drugs that have demonstrated potent antitumor activity and are currently been studied, such as Sacituzumab Govitecan (SG) and Datopotamab Deruxtecan (Dato-Dxd). In this study, we reviewed the progress of Trop-2 research in solid tumors. We also sorted out the composition and rationale of Trop-2 related drugs and summarized the related clinical trials. Finally, we discussed the current status of Trop-2 research and expanded our perspectives on its future research directions. Importantly, we found that Trop-2 targeted ADCs have great potential for combination with other antitumor therapies. Trop-2 targeted ADCs can reprogramme tumor microenvironment through multiple signaling pathways, ultimately activating antitumor immunity.     

Vascular Endothelial Growth Factor C-Induced Lymphangiogenesis Decreases Tumor Interstitial Fluid Pressure and Tumor

Characteristically, most solid tumors exhibit an increased tumor interstitial fluid pressure (TIFP) that directly contributes to the lowered uptake of macromolecular therapeutics into the tumor interstitium. Abnormalities in the tumor-associated lymph vessels are a central brick in the development and prolonged sustaining of an increased TIFP. In the current study, vascular endothelial growth factor C (VEGF-C) was used to enhance tumor-associated lymphangiogenesis as a new mechanism to actively reduce the TIFP by increased lymphatic drainage of the tumor tissue. Human A431 epidermoid vulva carcinoma cells were inoculated in NMRI nu/nu mice to generate a xenograft mouse model. Seven days after tumor cell injection, VEGF-C was peritumorally injected to induce lymphangiogenesis. Tumor growth and TIFP was lowered significantly over time in VEGF-C-treated tumors in comparison to control or VEGF-A-treated animals. These data demonstrate for the first time that actively induced lymphangiogenesis can lower the TIFP in a xenograft tumor model and apparently reduce tumor growth. This model represents a novel approach to modulate biomechanical properties of the tumor interstitium enabling a lowering of TIFP in vivo.     

Tet methylcytosine dioxygenase 1 promotes hypoxic gene induction and cell migration in colon cancer

Ten-eleven translocation 1 (TET1), a widely reported DNA demethylation protein, has been associated with tumorigenesis and metastasis. However, whether TET1 is an oncogene or tumor suppressor gene has been controversial; the mechanism of how TET1 affects cancer progression remains unclear. The current study aims to investigate how TET1 is changed in the tumor microenvironment and to explore the mechanisms of how TET1 affects colon cancer progression. Because hypoxia prevails on solid tumors, we established an important connection between hypoxia and DNA demethylation in tumorigenesis. By qPCR and RNA interference (RNAi) technology, we found that hypoxia increased TET1 expression with a hypoxia-inducible factor-1-alpha (HIF-1α)-dependent manner. By CHIP-qPCR and pyrosequencing technology, we demonstrated that TET1 regulated the target gene expression of HIF-1α through HIF-1α binding to hypoxia-responsive elements (HREs), and HIF-1α binding to HREs depended on CpG methylation levels. By Cell Counting Kit-8 (CCK-8) and transwell assay, we showed that loss of TET1 did not affect cell proliferation but inhibited migration. We also identified two novel gene mutants of TET1 in 120 paired tumor/normal tissue specimens by DNA sequencing and found that TET1 E2082K mutant blocked the TET1-enhanced cell migration. Our results showed that the downregulation of TET1 rescued the abnormally high levels of gene expression resulting from hypoxia in tumors and reduced the migration activity of tumor cells, suggesting a therapeutic role by interference with TET1 in colon cancer treatment. By demonstrating that hypoxia upregulated TET1 and that TET1 drove HIF-1α-responsive genes, we showed that an epigenetic mechanism and tumor microenvironment-driven models coexisted and mutually affected colon cancer.     

Inhibiting hypoxia-inducible factor 1 for cancer therapy

Hypoxia has long been recognized as a common feature of solid tumors and a negative prognostic factor for response to treatment and survival of cancer patients. The discovery of hypoxia-inducible factor 1 (HIF-1), a molecular determinant of the response of mammalian cells to hypoxia, has led to the identification of a "molecular target" of hypoxia suitable for the development of cancer therapeutics. Early controversy about whether or not HIF-1 is a good target for therapy has not discouraged academic groups and pharmaceutical companies from actively engaging in the discovery of small-molecule inhibitors of HIF. However, what is the best strategy to inhibit HIF and how HIF inhibitors should be developed for treatment of human cancers is still poorly defined. In this review, aspects related to the identification and early development of novel HIF inhibitors are discussed. Identification and validation of pharmacodynamic end points relevant to the HIF-1 pathway is essential for a rational development of HIF inhibitors. Integration of these biomarkers in early clinical trials may provide valuable information to determine the contribution of HIF inhibitors to response to therapy. Finally, HIF inhibitors should be incorporated in combination strategies to effectively target multiple cellular components of the tumor microenvironment and redundant signaling pathways frequently deregulated in human cancer.