Safeguard repressor locks hepatocyte identity and blocks liver cancer
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Active repression of cell fate plasticity by PROX1 safeguards hepatocyte identity and prevents liver tumorigenesis
Cell fate plasticity enables development, yet unlocked plasticity is a cancer hallmark. While transcription master regulators induce lineage-specific genes to restrict plasticity, it remains unclear whether plasticity is actively suppressed by lineage-specific repressors. Here we computationally predict so-called safeguard repressors for 18 cell types that block phenotypic plasticity lifelong. We validated hepatocyte-specific candidates using reprogramming, revealing that prospero homeobox protein 1 (PROX1) enhanced hepatocyte identity by direct repression of alternative fate master regulators. In mice, Prox1 was required for efficient hepatocyte regeneration after injury and was sufficient to prevent liver tumorigenesis. In line with patient data, Prox1 depletion caused hepatocyte fate loss in vivo and enabled the transition of hepatocellular carcinoma to cholangiocarcinoma. Conversely, overexpression promoted cholangiocarcinoma to hepatocellular carcinoma transdifferentiation. Our findings provide evidence for PROX1 as a hepatocyte-specific safeguard and support a model where cell-type-specific repressors actively suppress plasticity throughout life to safeguard lineage identity and thus prevent disease.
Collaborative orchestration of BH3-only proteins governs Bak/Bax-dependent hepatocyte apoptosis under antiapoptotic protein-deficiency in mice
The fine-tuned balance between anti-apoptotic Bcl-2 family proteins, such as Bcl-xL and Mcl-1, and pro-apoptotic Bcl-2 family proteins, like Bak and Bax, is crucial for maintaining hepatocyte integrity. BH3-only proteins, including Bid, Bim, Puma, Noxa, Bad, Bmf, Bik and Hrk, serve as apoptosis initiators. They are activated by various stimuli, which leads to Bak/Bax activation. We previously reported that Bid and Bim contributed to hepatocyte apoptosis through Bak/Bax activation in the absence of anti-apoptotic proteins Bcl-xL and/or Mcl-1. However, the comprehensive involvement of all eight BH3-only proteins in Bak/Bax-dependent hepatocyte apoptosis remains unclear. Puma disruption suppressed hepatocyte apoptosis in hepatocyte-specific Bcl-xL or Mcl-1 knockout (Bcl-xLΔHep/ΔHep or Mcl-1ΔHep/ΔHep) mice. Disruption of Bid and Bim partially prevented lethality in Mcl-1ΔHep/+ Bcl-xLΔHep/ΔHep mice, although severe hepatocyte apoptosis persisted, which was suppressed by additional Puma disruption. However, hepatocyte apoptosis was still induced compared to that in Mcl-1ΔHep/+ Bcl-xLΔHep/ΔHep BaxΔHep/ΔHep Bak−/− mice. Triple disruption of Bid, Bim and Puma did not prevent induction of hepatocyte apoptosis in tamoxifen-induced Mcl-1iΔHep/iΔHep Bcl-xLiΔHep/iΔHep mice. Primary hepatocytes, isolated from Mcl-1fl/fl Bcl-xLfl/fl Bid−/− Bim−/− Puma−/− mice and immortalized, underwent apoptosis with doxycycline-dependent Cre recombination. Among the remaining five BH3-only proteins, Bik and Hrk were not expressed in these cells, and Noxa knockdown, but not Bad or Bmf knockdown, reduced apoptosis. Noxa disruption alleviated hepatocyte apoptosis in Mcl-1ΔHep/ΔHep mice and tamoxifen-induced Mcl-1iΔHep/iΔHep Bcl-xLiΔHep/iΔHep Bid−/− Bim−/− Puma−/− mice, prolonging survival. Apoptosis persisted in immortalized primary hepatocytes isolated from Mcl-1fl/fl Bcl-xLfl/fl Bid−/− Bim−/− Puma−/− Noxa−/− mice where doxycycline-dependent Cre recombination was induced, but was completely suppressed by Bak/Bax knockdown, while Bad or Bmf knockdown had no effect. In conclusion, among the eight BH3-only proteins, Puma and Noxa, alongside Bid and Bim, contributed to Bak/Bax-dependent hepatocyte apoptosis, but not indispensably, in the absence of Mcl-1 and Bcl-xL.
AAV capsid prioritization in normal and steatotic human livers maintained by machine perfusion
Therapeutic efficacy and safety of adeno-associated virus (AAV) liver gene therapy depend on capsid choice. To predict AAV capsid performance under near-clinical conditions, we established side-by-side comparison at single-cell resolution in human livers maintained by normothermic machine perfusion. AAV-LK03 transduced hepatocytes much more efficiently and specifically than AAV5, AAV8 and AAV6, which are most commonly used clinically, and AAV-NP59, which is better at transducing human hepatocytes engrafted in immune-deficient mice. AAV-LK03 preferentially transduced periportal hepatocytes in normal liver, whereas AAV5 targeted pericentral hepatocytes in steatotic liver. AAV5 and AAV8 transduced liver sinusoidal endothelial cells as efficiently as hepatocytes. AAV capsid and steatosis influenced vector episome formation, which determines gene therapy durability, with AAV5 delaying concatemerization. Our findings inform capsid choice in clinical AAV liver gene therapy, including consideration of disease-relevant hepatocyte zonation and effects of steatosis, and facilitate the development of AAV capsids that transduce hepatocytes or other therapeutically relevant cell types in the human liver with maximum efficiency and specificity.
Enhancer reprogramming: critical roles in cancer and promising therapeutic strategies
Transcriptional dysregulation is a hallmark of cancer initiation and progression, driven by genetic and epigenetic alterations. Enhancer reprogramming has emerged as a pivotal driver of carcinogenesis, with cancer cells often relying on aberrant transcriptional programs. The advent of high-throughput sequencing technologies has provided critical insights into enhancer reprogramming events and their role in malignancy. While targeting enhancers presents a promising therapeutic strategy, significant challenges remain. These include the off-target effects of enhancer-targeting technologies, the complexity and redundancy of enhancer networks, and the dynamic nature of enhancer reprogramming, which may contribute to therapeutic resistance. This review comprehensively encapsulates the structural attributes of enhancers, delineates the mechanisms underlying their dysregulation in malignant transformation, and evaluates the therapeutic opportunities and limitations associated with targeting enhancers in cancer.
Targeting of TAMs: can we be more clever than cancer cells?
With increasing incidence and geography, cancer is one of the leading causes of death, reduced quality of life and disability worldwide. Principal progress in the development of new anticancer therapies, in improving the efficiency of immunotherapeutic tools, and in the personification of conventional therapies needs to consider cancer-specific and patient-specific programming of innate immunity. Intratumoral TAMs and their precursors, resident macrophages and monocytes, are principal regulators of tumor progression and therapy resistance. Our review summarizes the accumulated evidence for the subpopulations of TAMs and their increasing number of biomarkers, indicating their predictive value for the clinical parameters of carcinogenesis and therapy resistance, with a focus on solid cancers of non-infectious etiology. We present the state-of-the-art knowledge about the tumor-supporting functions of TAMs at all stages of tumor progression and highlight biomarkers, recently identified by single-cell and spatial analytical methods, that discriminate between tumor-promoting and tumor-inhibiting TAMs, where both subtypes express a combination of prototype M1 and M2 genes. Our review focuses on novel mechanisms involved in the crosstalk among epigenetic, signaling, transcriptional and metabolic pathways in TAMs. Particular attention has been given to the recently identified link between cancer cell metabolism and the epigenetic programming of TAMs by histone lactylation, which can be responsible for the unlimited protumoral programming of TAMs. Finally, we explain how TAMs interfere with currently used anticancer therapeutics and summarize the most advanced data from clinical trials, which we divide into four categories: inhibition of TAM survival and differentiation, inhibition of monocyte/TAM recruitment into tumors, functional reprogramming of TAMs, and genetic enhancement of macrophages.
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