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Intrabody Targeting HIF-1α Mediates Transcriptional Downregulation of Target Genes Related to Solid Tumors.

ABSTRACT: Uncontrolled growth of solid tumors will result in a hallmark hypoxic condition, whereby the key transcriptional regulator of hypoxia inducible factor-1α (HIF-1α) will be stabilized to activate the transcription of target genes that are responsible for the metabolism, proliferation, and metastasis of tumor cells. Targeting and inhibiting the transcriptional activity of HIF-1 may provide an interesting strategy for cancer therapy. In the present study, an immune library and a synthetic library were constructed for the phage display selection of Nbs against recombinant PAS B domain protein (rPasB) of HIF-1α. After panning and screening, seven different nanobodies (Nbs) were selected, of which five were confirmed via immunoprecipitation to target the native HIF-1α subunit. The inhibitory effect of the selected Nbs on HIF-1 induced activation of target genes has been evaluated after intracellular expression of these Nbs in HeLa cells. The dramatic inhibition of both intrabody formats on the expression of HIF-1-related target genes has been confirmed, which indicated the inhibitory efficacy of selected Nbs on the transcriptional activity of HIF-1.

SUBMITTER: Hu Y

PROVIDER: S-EPMC8625554 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Intrabody Targeting HIF-1α Mediates Transcriptional Downregulation of Target Genes Related to Solid Tumors.

Hu Yaozhong Y Romão Ema E Vincke Cécile C Brys Lea L Elkrim Yvon Y Vandevenne Marylène M Liu Changxiao C Muyldermans Serge S

International journal of molecular sciences 20211115 22

Uncontrolled growth of solid tumors will result in a hallmark hypoxic condition, whereby the key transcriptional regulator of hypoxia inducible factor-1α (HIF-1α) will be stabilized to activate the transcription of target genes that are responsible for the metabolism, proliferation, and metastasis of tumor cells. Targeting and inhibiting the transcriptional activity of HIF-1 may provide an interesting strategy for cancer therapy. In the present study, an immune library and a synthetic library we ...[more]

PMID: 34830219

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Project description:The hypoxic tumor microenvironment is the major contributor of chemotherapy resistance in solid tumors. One of the key regulators of hypoxic responses within the cell is the hypoxia inducible factor-1α (HIF-1α) that is involved in transcription of genes promoting cell survival and chemotherapy resistance. Multidrug resistance gene-1 (MDR1) and Lysosome-associated protein transmembrane 4B-35 (LAPTM4B-35) are among those notable players which augment their responses to cellular hypoxia. MDR1 is the hypoxia responsive gene involved in multidrug resistance phenotype while LAPTM4B-35 is involved in chemotherapy resistance by stabilizing HIF-1α and overexpressing MDR1. Overexpression of HIF-1α, MDR1 and LAPTM4B has been associated with poor disease outcome in many cancers when studied individually at tissue level. However, accessibility of the tissues following the course of chemotherapy for ascertaining chemotherapy resistance is difficult and sometimes not clinically feasible. Therefore, indication of hypoxic biomarkers in patient's blood can significantly alter the clinical outcome. Hence there is a need to identify a blood based marker to understand the disease progression. In the current study the expression of hypoxia associated chemotherapy resistance genes were studied in the peripheral blood lymphocytes of solid tumor patients and any potential correlation with disease progression were explored. The expression of HIF-1α, MDR1 and LAPTM4B was studied in blood of 72 breast, 42 ovarian, 32 colon and 21 prostate cancer patients through real time PCR analysis using delta cycle threshold method. The statistical scrutiny was executed through Fisher's Exact test and the Spearman correlation method. There was 12-13 fold increased in expression of HIF-1α, two fold increased in MDR1 and 13-14 fold increased in LAPTM4B mRNA level in peripheral blood of breast, ovarian, prostate and colon cancer patients. In the current study there was an association of HIF-1α, MDR1 and LAPTM4B expression with advanced tumor stage, metastasis and chemotherapy treated group in breast, ovarian, prostate and colon cancer patients. The Spearman analysis also revealed a positive linear association among HIF-1α, MDR1 and LAPTM4B in all the studied cancer patients. The elevated expression of HIF-1α, MDR1 and LAPTM4B in peripheral blood of solid tumor patients can be a predictor of metastasis, disease progression and treatment response in these cancers. However, larger studies are needed to further strengthen their role as a potential biomarker for cancer prognosis.

Project description:BackgroundProstate cancer (PCa) ranks as the second most prevalent cancer in men, with advanced stages posing significant treatment challenges. Given its solid tumor nature, PCa is highly susceptible to hypoxia, a condition associated with resistance to radiation and chemotherapy, metastasis, and unfavorable patient outcomes. Hypoxia-inducible factors (HIFs) play a pivotal role in cancer cell adaptation to hypoxic environments, contributing to treatment resistance. Consequently, inhibitors targeting HIFs hold promise for cancer therapy.MethodsIn this study, we aimed to characterize novel HIF-1α inhibitors including Sodwanones A (1), B (2), C (3), G (4) and Yardenone 2 (5) isolated from marine sponges belonging to the Axinella genus. Our investigation evaluated the impact of these compounds on various aspects of HIF-1α regulation, including stabilization, nuclear localization, expression of HIF-1 target genes (while sparing HIF-2 target genes), cellular metabolism, as well as cell proliferation and viability in prostate cells under hypoxic conditions.ResultsOur findings revealed that among the compounds tested, Yardenone 2 exhibited notable effects in hypoxia: it destabilized HIF-1α at the protein level, decreased its nuclear localization, selectively altered the expression of HIF-1 target genes, and restrained cell proliferation in aggressive PC3 prostate cancer cells as well as in an MSK-PCa3 patient-derived organoid line. Moreover, it affected the morphology of these organoid. Yardenone 2 was also compared to Docetaxel, a specific microtubule inhibitor and a drug used in the treatment of prostate cancer. The comparison between the two compounds revealed notable differences, such as a lack of specificity to hypoxic cells of Docetaxel.ConclusionThese results mark the first demonstration that Yardenone 2 functions as a cytostatic-like inhibitor impacting microtubules, specifically targeting hypoxic cancer cells. This discovery suggests a promising avenue for novel therapeutic interventions in prostate cancer.

Project description:BackgroundEpithelial-mesenchymal transition (EMT) of mesothelial cells is a key step in the peritoneal fibrosis (PF). Recent evidence indicates that signal transducer and activator of transcription 3 (STAT3) might mediate the process of renal fibrosis, which could induce the expression of hypoxia-inducible factor-1α (HIF-1α). Here, we investigated the effect of STAT3 activation on HIF-1α expression and the EMT of mesothelial cells, furthermore the role of pharmacological blockade of STAT3 in the process of PF during peritoneal dialysis (PD) treatment.MethodsFirstly, we investigated the STAT3 signaling in human peritoneal mesothelial cells (HPMCs) from drained PD effluent. Secondly, we explored the effect of STAT3 signaling activation on the EMT and the expression of HIF-1α in human mesothelial cells (Met-5A) induced by high glucose. Finally, peritoneal fibrosis was induced by daily intraperitoneal injection with peritoneal dialysis fluid (PDF) so as to explore the role of pharmacological blockade of STAT3 in this process.ResultsCompared with the new PD patient, the level of phosphorylated STAT3 was up-regulated in peritoneal mesothelial cells from long-term PD patients. High glucose (60 mmol/L) induced over-expression of Collagen I, Fibronectin, α-SMA and reduced the expression of E-cadherin in Met-5A cells, which could be abrogated by STAT3 inhibitor S3I-201 pretreatment as well as by siRNA for STAT3. Furthermore, high glucose-mediated STAT3 activation in mesothelial cells induced the expression of HIF-1α and the profibrotic effect of STAT3 signaling was alleviated by siRNA for HIF-1α. Daily intraperitoneal injection of high-glucose based dialysis fluid (HG-PDF) induced peritoneal fibrosis in the mice, accompanied by the phosphorylation of STAT3. Immunostaining showed that phosphorylated STAT3 was expressed mostly in α-SMA positive cells in the peritoneal membrane induced by HG-PDF. Administration of S3I-201 prevented the progression of peritoneal fibrosis, angiogenesis, macrophage infiltration as well as the expression of HIF-1α in the peritoneal membrane induced by high glucose.ConclusionsTaken together, these findings identified a novel mechanism linking STAT3/HIF-1α signaling to peritoneal fibrosis during long-term PD treatment. It provided the first evidence that pharmacological inhibition of STAT3 signaling attenuated high glucose-mediated mesothelial cells EMT as well as peritoneal fibrosis.

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Intrabody Targeting HIF-1α Mediates Transcriptional Downregulation of Target Genes Related to Solid Tumors.

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Intrabody Targeting HIF-1α Mediates Transcriptional Downregulation of Target Genes Related to Solid Tumors.

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