A Study Of Roles And Mechanisms Of IASPP In Regulating HIF-1α Activity Under Normoxia In Tumor Cells

Rapid proliferation is one of the hallmark characteristics of tumors.Due to its rapid growth and relatively insufficient angiogenesis,tumor tissue is often in a state of hypoxia.Hypoxia-inducible factor 1-alpha(HIF-1α)is a major regulator of cellular adaptation to hypoxia,and it is highly expressed in a variety of tumor tissues.However,its activation state is not always associated with hypoxia state.It is worth noting that under normoxic conditions,the activation of HIF-1α can lead to a pseudohypoxic response in cells,and a large number of downstream target genes of HIF are activated,leading to metabolic reprogramming of tumor cells and promoting tumorigenesis and development.Therefore,revealing the activation mechanism of HIF-1α under normoxic conditions can provide new clues for in-depth understanding of tumor pathological characteristics and pathogenesis,and is expected to promote the development of new anti-cancer programs.Inhibitor of Apoptosis Stimulating Protein of p53(iASPP)is an important inhibitor of apoptosis,which can bind to p53 in the nucleus and exert a tumorpromoting effect by inhibiting the activity of p53.However,iASPP is mainly localized in the cytoplasm and can stabilize and activate nuclear factor E2-related factor 2(Nrf2)in a p53-independent mechanism to inhibit apoptosis and promote drug resistance.However,it remains unclear whether this gene can affect other tumor-specific changes beyond apoptosis tolerance.In this paper,the bioinformatics analysis of RNA-seq data of tumor cells with different expression levels of iASPP found that the expression level of iASPP was related to the HIF signaling pathway,suggesting that iASPP may affect the characteristic changes of HIF-related tumors.Afterwards,this paper investigates whether iASPP is involved in the activation of HIF-1α signaling.The study found that although the expression levels of iASPP under normoxic and hypoxic conditions were similar,iASPP could regulate the transcriptional activity of HIF-1α and the expression of target genes related to angiogenesis and metabolism under both normoxic and hypoxic conditions.Significantly promoted HIF-1α-dependent angiogenesis and glycolysis,while HIF-1α knockdown completely inhibited the above-mentioned effects of iASPP.On this basis,this paper further explores the molecular mechanism of iASPP regulating HIF-1α signaling pathway.The results showed that iASPP had no significant effect on the m RNA level of HIF-1α,while proteasome inhibitors could significantly inhibit the regulatory effect of iASPP on HIF-1α,suggesting that iASPP could increase the expression of HIF-1α by inhibiting the HIF-1α ubiquitin-proteasome pathway.The half-life of HIF-1α under normoxia is about 5 minutes.By optimizing the classical protein detection technology,this paper realized the detection of HIF-1α level under normoxia.Based on this,the molecular mechanism of iASPP’s regulation of HIF-1αwas discovered: iASPP nitrogen-terminal sequence(1-294)contains a key VHL binding site,and this sequence fragment competes with HIF-1α to bind VHL(Y98),which weakens the binding of HIF-1α to VHL and VHL-mediated degradation of HIF-1α by ubiquitination lead to increased HIF-1α protein stability and activity.To understand the role of the iASPP-HIF-1α axis in vivo,we examined the expression levels of iASPP and HIF-1α in colon cancer tissues.The results showed that iASPP was positively correlated with the protein level of HIF-1α.The regulatory effect of iASPP on HIF-1α was further verified in a nude mouse tumorigenesis model.Knockdown of iASPP significantly inhibited the growth of subcutaneously transplanted tumors in nude mice.However,compared with the control group,although the volume of iASPP knockdown tumors was smaller,the intratumoral necrosis area was significantly higher than that of the control group,suggesting that iASPP may play an important regulation role in the ability of tumor tissue to tolerate hypoxia in addition to inhibiting apoptosis.Further experimental studies showed that inhibiting the expression of HIF-1α could produce a tumor-suppressive effect similar to iASPP knockdown,and could greatly limit the effect of iASPP knockdown,suggesting that iASPP may play a tumor-promoting effect through HIF-1α.The study also found that iASPP knockdown can significantly improve the sensitivity of colorectal cancer xenografts to the HIF-1αinhibitor YC-1,so that YC-1 can play an important tumor suppressor effect at low concentrations.Therefore,the research and development of iASPP inhibitors and their combination therapy with chemotherapeutic drugs provide more solutions and basis for conquering tumors and improving tumor treatment effects,suggesting that the iASPPHIF-1α axis is promising as an important new target for anticancer therapy.In conclusion,this paper reveals that iASPP regulates the continuous activation of HIF-1α in tumor cells under normoxic conditions and the molecular mechanism,provides a mechanistic explanation for the pseudo-hypoxic response of tumor cells,and provides a mechanism for targeting HIF-1α to inhibit tumors.It provides a new target and also provides important clues for the therapeutic effect of HIF-1α inhibitors.