Transcriptomics and proteomics are potent research methods employed to delve deeply into alterations in gene expression and protein levels within organisms. In cancer research, the joint utilization of these techniques unveils the pivotal regulatory function of hypoxia-inducible factors in tumor onset and progression.
Hypoxia is prevalent in the tumor microenvironment, where cells often thrive in oxygen-deprived surroundings. This hypoxic state triggers specific gene activation, leading to the synthesis of proteins that facilitate cellular adaptation and response to low oxygen conditions. Among these, the Hypoxia-Inducible Factor (HIF) family stands out as vital regulatory agents governing diverse tumor-related processes such as angiogenesis, cell proliferation, and metabolic reconfiguration.
Employing transcriptomics, researchers can contrast gene expression patterns in tumor and normal cells under distinct hypoxic states. This enables the identification of hypoxia-driven gene modulation and deepens our comprehension of their roles in tumor development. In parallel, proteomics technology unveils alterations in protein expression levels and modifications, offering insights into hypoxia's impact on protein regulation.
Thoroughly analyzing transcriptomic and proteomic data empowers researchers to unravel signaling pathways and molecular mechanisms linked to hypoxia-inducible factors. This, in turn, sheds light on the specific contribution of these factors to tumor cell adaptation to oxygen-depleted environments and the facilitation of tumor growth and dissemination. These findings not only enhance our grasp of tumorigenesis mechanisms but also inspire novel therapeutic strategies targeting hypoxia-inducible factors.
Case. Transcriptomic and Proteomic Analyses Provide Comprehensive Insights into the Regulatory Role of Hypoxia-Inducible Factors in Tumorigenesis.
The biallelic inactivation of the Von Hippel-Lindau (VHL) tumor suppressor gene is a crucial genetic event in the majority of clear cell renal cell carcinomas (ccRCC). This event collaborates with mutations or chromosomal copy number alterations in certain core epigenetic regulatory genes (including PBRM1, BAP1, SETD2, and KDM5C), cell cycle control genes (such as TP53, CDKN2A, and MYC), or PI3K pathway genes (including PIK3CA, PTEN, MTOR, and TSC1) to collectively promote the progression of ccRCC tumors. This viewpoint is supported by numerous murine models that demonstrate these gene interactions.
To investigate the roles of Hif1α and Hif2α in the development of ccRCC, a mouse model of ccRCC was employed, utilizing tamoxifen-induced renal epithelial cell-specific deletion (Ksp-CreERT2) of Vhl, Trp53, and Rb1. This ccRCC mouse model, which partially recapitulates many features of human ccRCC progression, was utilized to study the functions of Hif1α and Hif2α in ccRCC.
Phenotypic Study of Hif1α and Hif2α Deletions: Strong Dependency of ccRCC Formation on Hif1α
The authors first generated mouse models with different gene deletions:
(1) VhlΔ/ΔTrp53Δ/ΔRb1Δ/Δ
(2) VhlΔ/ΔTrp53Δ/ΔRb1Δ/ΔHif1αΔ/Δ
(3) VhlΔ/ΔTrp53Δ/ΔRb1Δ/ΔHif2αΔ/Δ
(4) ΔTrp53Δ/ΔRb1Δ/Δ
Tumor formation in each model was assessed, revealing that VHL deficiency promoted tumor initiation and growth in the ΔTrp53Δ/ΔRb1Δ/Δ background. Hif1α deletion completely reversed the pro-tumorigenic effect of VHL deficiency. Hif2α deletion led to moderate tumor development. No significant changes in tumor metastasis were observed in any of the genotypic mouse models. These data indicate that Hif1α is crucial for the effective progression and growth of Vhl-mutated ccRCCs, whereas Hif2α is only partially essential.
Hypoxia-Inducible Factor-1α (Hif1α) Exerts Effects Only in the Primary Cancer Model
The authors initially obtained embryonic fibroblasts of different genotypes in mice. Following deletion of Vhl and/or Hif1α and/or Hif2α, the proliferative capacity of the cells was assessed. The results indicated that Vhl deficiency led to a decrease in proliferative potential. Interestingly, in contrast to other research findings, in immortalized Vhlfl/flTrp53fl/fl mouse embryonic fibroblasts, the loss of proliferative capacity was found to be Hif1α-dependent. This suggests that in the absence of Vhl, Hif1α plays a role in inhibiting cell proliferation.
Subsequently, ccRCC cells were obtained from VhlΔ/ΔTrp53Δ/ΔRb1Δ/Δ mice, and the proliferative capacity of the cells was tested through the re-expression of pVHL or deletion of Hif1α. The results demonstrated that neither the re-expression of pVHL nor the absence of Hif1α affected cell proliferation, but both significantly promoted cell growth. Furthermore, using a syngeneic tumor transplantation model, it was confirmed that pVhl re-expression significantly delayed tumor growth, whereas Hif1α knockdown did not.
Taken together, these findings indicate that Hif1α inhibits the proliferation of normal mouse cells lacking VHL, but it does not impact the proliferation of mouse ccRCC cells or the formation of syngeneic transplanted tumors. This highlights the specific requirement of Hif1α for primary tumor initiation. It also suggests that Hif1α exerts a significant oncogenic effect only within the physiological environment of renal epithelium.
Transcriptomic Analysis: Effects of Hif1α and Hif2α on Mouse ccRCC Transcriptomes
The authors conducted RNA sequencing on samples including WT cortex, Vhl & Trp53 & Rb1 triple-deficient tumor, Vhl & Trp53 & Rb1 & Hif1α quadruple-deficient tumor, and Vhl & Trp53 & Rb1 & Hif2α quadruple-deficient tumor. PCA analysis revealed significant transcriptional differences between the WT samples and the other samples. The analysis indicated that HIF-1α regulates glycolytic processes, while HIF-2α is involved in the regulation of genes related to lipoprotein metabolism, ribosome biogenesis, as well as the transcriptional activity of E2F and MYC. Interestingly, genes upregulated in Hif2α-deficient tumor tissues include those associated with interferon signaling, T cell activation, innate and adaptive immunity, antigen processing and presentation, NF-κB-related genes, and IRF transcription factor target genes, suggesting alterations in the tumor's immune environment.
Proteomic Analysis: Effects of Hif1α and Hif2α on Mouse ccRCC Proteome
In a further step, the researchers conducted comprehensive proteomic profiling of samples, including wild-type (WT) cortical tissue, Vhl & Trp53 & Rb1 triple-deficient tumor samples, Vhl & Trp53 & Rb1 & Hif1α quadruple-deficient tumor samples, and Vhl & Trp53 & Rb1 & Hif2α quadruple-deficient tumor samples. This proteomic analysis quantified a total of 4257 proteins. Comparatively, the global transcriptomic and proteomic comparisons demonstrated relatively low correlation, underscoring the distinct layers of biological information provided by each approach.
The findings of differential protein expression enrichment mirrored those observed in the transcriptomic analysis. Specifically, the loss of HIF1α was associated with a reduction in the expression of glycolytic enzymes, while it concomitantly led to an increase in the expression of proteins linked to oxidative phosphorylation and respiratory electron transport. In contrast, Hif2α deficiency resulted in a decrease in the expression of genes targeted by the MYC transcription factor. Consequently, this deficiency led to an elevation in the expression of genes associated with immune responses, interferon signaling, cytokine signaling, and antigen presentation.
In summary, the comparative analyses of the proteomic and transcriptomic data reinforce and validate each other, enhancing our understanding of the distinct biological roles played by Hif1α and Hif2α in the context of ccRCC. These parallel analyses serve as an independent validation of the biological disparities between different tumor genotypes, providing a more comprehensive perspective on the intricate molecular mechanisms underlying ccRCC progression.
Hif2α Deletion Alters Antigen Presentation in ccRCC
Both transcriptomic and proteomic analyses revealed an upregulation of antigen presentation-related genes in the tumor models of mice lacking Hif2α. This included elevated expression of major histocompatibility complex (MHC) class I and class II genes, as well as genes associated with antigen presentation. By analyzing TCGA mRNA sequencing data, the authors confirmed the widespread expression of Hif2α in tumors.
Subsequently, using Pearson correlation analysis, the authors found a negative correlation between Hif2α and three out of six MHC class I genes, twelve out of fifteen MHC class I genes, and twenty-six out of fifty-one non-MHC genes related to antigen presentation. Combining the results from the mouse models and the human TCGA database, the authors conclude that Hif2α inhibits antigen presentation.
Reference
- Hoefflin, Rouven, et al. "HIF-1α and HIF-2α differently regulate tumour development and inflammation of clear cell renal cell carcinoma in mice." Nature communications 11.1 (2020): 4111.
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