TERT activation targets DNA methylation and multiple aging hallmarks.

Insufficient telomerase activity, stemming from low telomerase reverse transcriptase (TERT) gene transcription, contributes to telomere dysfunction and aging pathologies. Besides its traditional function in telomere synthesis, TERT acts as a transcriptional co-regulator of genes pivotal in aging and age-associated diseases. Here, we report the identification of a TERT activator compound (TAC) that upregulates TERT transcription via the MEK/ERK/AP-1 cascade. In primary human cells and naturally aged mice, TAC-induced elevation of TERT levels promotes telomere synthesis, blunts tissue aging hallmarks with reduced cellular senescence and inflammatory cytokines, and silences p16INK4a expression via upregulation of DNMT3B-mediated promoter hypermethylation. In the brain, TAC alleviates neuroinflammation, increases neurotrophic factors, stimulates adult neurogenesis, and preserves cognitive function without evident toxicity, including cancer risk. Together, these findings underscore TERT's critical role in aging processes and provide preclinical proof of concept for physiological TERT activation as a strategy to mitigate multiple aging hallmarks and associated pathologies.

Antitelomerase therapy provokes ALT and mitochondrial adaptive mechanisms in cancer.

To assess telomerase as a cancer therapeutic target and determine adaptive mechanisms to telomerase inhibition, we modeled telomerase reactivation and subsequent extinction in T cell lymphomas arising in Atm(-/-) mice engineered with an inducible telomerase reverse transcriptase allele. Telomerase reactivation in the setting of telomere dysfunction enabled full malignant progression with alleviation of telomere dysfunction-induced checkpoints. These cancers possessed copy number alterations targeting key loci in human T cell lymphomagenesis. Upon telomerase extinction, tumor growth eventually slowed with reinstatement of telomere dysfunction-induced checkpoints, yet growth subsequently resumed as tumors acquired alternative lengthening of telomeres (ALT) and aberrant transcriptional networks centering on mitochondrial biology and oxidative defense. ALT+ tumors acquired amplification/overexpression of PGC-1ß, a master regulator of mitochondrial biogenesis and function, and they showed marked sensitivity to PGC-1ß or SOD2 knockdown. Genetic modeling of telomerase extinction reveals vulnerabilities that motivate coincidental inhibition of mitochondrial maintenance and oxidative defense mechanisms to enhance antitelomerase cancer therapy.

Oncogenic Kras drives invasion and maintains metastases in colorectal cancer.

Human colorectal cancer (CRC) is a major cause of cancer mortality and frequently harbors activating mutations in the KRAS gene. To understand the role of oncogenic KRAS in CRC, we engineered a mouse model of metastatic CRC that harbors an inducible oncogenic Kras allele (Krasmut ) and conditional null alleles of Apc and Trp53 (iKAP). The iKAP model recapitulates tumor progression from adenoma through metastases. Whole-exome sequencing revealed that the Krasmut allele was heterogenous in primary tumors yet homogenous in metastases, a pattern consistent with activated Krasmut signaling being a driver of progression to metastasis. System-level and functional analyses revealed the TGF-ß pathway as a key mediator of Krasmut -driven invasiveness. Genetic extinction of Krasmut resulted in specific elimination of the Krasmut subpopulation in primary and metastatic tumors, leading to apoptotic elimination of advanced invasive and metastatic disease. This faithful CRC model provides genetic evidence that Krasmut drives CRC invasion and maintenance of metastases.

Epidermal sensing of oxygen is essential for systemic hypoxic response.

Skin plays an essential role, mediated in part by its remarkable vascular plasticity, in adaptation to environmental stimuli. Certain vertebrates, such as amphibians, respond to hypoxia in part through the skin; but it is unknown whether this tissue can influence mammalian systemic adaptation to low oxygen levels. We have found that epidermal deletion of the hypoxia-responsive transcription factor HIF-1alpha inhibits renal erythropoietin (EPO) synthesis in response to hypoxia. Conversely, mice with an epidermal deletion of the von Hippel-Lindau (VHL) factor, a negative regulator of HIF, have increased EPO synthesis and polycythemia. We show that nitric oxide release induced by the HIF pathway acts on cutaneous vascular flow to increase systemic erythropoietin expression. These results demonstrate that in mice the skin is a critical mediator of systemic responses to environmental oxygen.

HIF isoforms in the skin differentially regulate systemic arterial pressure.

Vascular flow through tissues is regulated via a number of homeostatic mechanisms. Localized control of tissue blood flow, or autoregulation, is a key factor in regulating tissue perfusion and oxygenation. We show here that the net balance between two hypoxia-inducible factor (HIF) transcription factor isoforms, HIF-1α and HIF-2α, is an essential mechanism regulating both local and systemic blood flow in the skin of mice. We also show that balance of HIF isoforms in keratinocyte-specific mutant mice affects thermal adaptation, exercise capacity, and systemic arterial pressure. The two primary HIF isoforms achieve these effects in opposing ways that are associated with HIF isoform regulation of nitric oxide production. We also show that a correlation exists between altered levels of HIF isoforms in the skin and the degree of idiopathic hypertension in human subjects. Thus, the balance between HIF-1α and HIF-2α expression in keratinocytes is a control element of both tissue perfusion and systemic arterial pressure, with potential implications in human hypertension.

KRAS-IRF2 Axis Drives Immune Suppression and Immune Therapy Resistance in Colorectal Cancer.

The biological functions and mechanisms of oncogenic KRASG12D (KRAS∗) in resistance to immune checkpoint blockade (ICB) therapy are not fully understood. We demonstrate that KRAS∗ represses the expression of interferon regulatory factor 2 (IRF2), which in turn directly represses CXCL3 expression. KRAS∗-mediated repression of IRF2 results in high expression of CXCL3, which binds to CXCR2 on myeloid-derived suppressor cells and promotes their migration to the tumor microenvironment. Anti-PD-1 resistance of KRAS∗-expressing tumors can be overcome by enforced IRF2 expression or by inhibition of CXCR2. Colorectal cancer (CRC) showing higher IRF2 expression exhibited increased responsiveness to anti-PD-1 therapy. The KRAS∗-IRF2-CXCL3-CXCR2 axis provides a framework for patient selection and combination therapies to enhance the effectiveness of ICB therapy in CRC.

MAPRE1 as a plasma biomarker for early-stage colorectal cancer and adenomas.

Blood-based biomarkers for early detection of colorectal cancer could complement current approaches to colorectal cancer screening. We previously identified the APC-binding protein MAPRE1 as a potential colorectal cancer biomarker. Here, we undertook a case-control validation study to determine the performance of MAPRE1 in detecting early colorectal cancer and colon adenoma and to assess the potential relevance of additional biomarker candidates. We analyzed plasma samples from 60 patients with adenomas, 30 with early colorectal cancer, 30 with advanced colorectal cancer, and 60 healthy controls. MAPRE1 and a set of 21 proteins with potential biomarker utility were assayed using high-density antibody arrays, and carcinoembryonic antigen (CEA) was assayed using ELISA. The biologic significance of the candidate biomarkers was also assessed in colorectal cancer mouse models. Plasma MAPRE1 levels were significantly elevated in both patients with adenomas and patients with colorectal cancer compared with controls (P < 0.0001). MAPRE1 and CEA together yielded an area under the curve of 0.793 and a sensitivity of 0.400 at 95% specificity for differentiating early colorectal cancer from controls. Three other biomarkers (AK1, CLIC1, and SOD1) were significantly increased in both adenoma and early colorectal cancer patient plasma samples and in plasma from colorectal cancer mouse models at preclinical stages compared with controls. The combination of MAPRE1, CEA, and AK1 yielded sensitivities of 0.483 and 0.533 at 90% specificity and sensitivities of 0.350 and 0.467 at 95% specificity for differentiating adenoma and early colorectal cancer, respectively, from healthy controls. These findings suggest that MAPRE1 can contribute to the detection of early-stage colorectal cancer and adenomas together with other biomarkers.

Genomic sequencing of colorectal adenocarcinomas identifies a recurrent VTI1A-TCF7L2 fusion.

Prior studies have identified recurrent oncogenic mutations in colorectal adenocarcinoma and have surveyed exons of protein-coding genes for mutations in 11 affected individuals. Here we report whole-genome sequencing from nine individuals with colorectal cancer, including primary colorectal tumors and matched adjacent non-tumor tissues, at an average of 30.7× and 31.9× coverage, respectively. We identify an average of 75 somatic rearrangements per tumor, including complex networks of translocations between pairs of chromosomes. Eleven rearrangements encode predicted in-frame fusion proteins, including a fusion of VTI1A and TCF7L2 found in 3 out of 97 colorectal cancers. Although TCF7L2 encodes TCF4, which cooperates with ß-catenin in colorectal carcinogenesis, the fusion lacks the TCF4 ß-catenin-binding domain. We found a colorectal carcinoma cell line harboring the fusion gene to be dependent on VTI1A-TCF7L2 for anchorage-independent growth using RNA interference-mediated knockdown. This study shows previously unidentified levels of genomic rearrangements in colorectal carcinoma that can lead to essential gene fusions and other oncogenic events.

The glial cell response is an essential component of hypoxia-induced erythropoiesis in mice.

A key adaptation to environmental hypoxia is an increase in erythropoiesis, driven by the hormone erythropoietin (EPO) through what is traditionally thought to be primarily a renal response. However, both neurons and astrocytes (the largest subpopulation of glial cells in the CNS) also express EPO following ischemic injury, and this response is known to ameliorate damage to the brain. To investigate the role of glial cells as a component of the systemic response to hypoxia, we created astrocyte-specific deletions of the murine genes encoding the hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha and their negative regulator von Hippel-Lindau (VHL) as well as astrocyte-specific deletion of the HIF target gene Vegf. We found that loss of the hypoxic response in astrocytes does not cause anemia in mice but is necessary for approximately 50% of the acute erythropoietic response to hypoxic stress. In accord with this, erythroid progenitor cells and reticulocytes were substantially reduced in number in mice lacking HIF function in astrocytes following hypoxic stress. Thus, we have demonstrated that the glial component of the CNS is an essential component of hypoxia-induced erythropoiesis.

Critical role of HIF-1alpha in keratinocyte defense against bacterial infection.

Skin, the first barrier against invading microorganisms, is hypoxic, even under baseline conditions. The transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha, the principal regulator of cellular adaptation to low oxygen, is strongly expressed in skin epithelium. HIF-1alpha is now understood to play a key role in the bactericidal capacity of phagocytic cells such as macrophages and neutrophils. In the skin, keratinocytes provide a direct antibacterial activity through production of antimicrobial peptides, including cathelicidin. Here, we generate mice with a keratinocyte-specific deletion of HIF-1alpha and examine effects on intrinsic skin immunity. Keratinocyte HIF-1alpha is seen to provide protection against necrotic skin lesions produced by the pathogen group A Streptococcus. RNA interference studies reveal that HIF-1alpha regulation of keratinocyte cathelicidin production is critical to their antibacterial function.

Waiting to inhale: HIF-1 modulates aerobic respiration.

The hypoxia-inducible factor HIF-1 is known to promote anaerobic respiration during low oxygen conditions (hypoxia). In this issue, Fukuda et al. (2007) expand the range of HIF-1's functions by showing that it modulates aerobic respiration as well.

Nucleotide sequence analysis of the enteropathogenic Escherichia coli adherence factor plasmid pMAR7.

The complete nucleotide sequence was determined for pMAR7, an enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid that contains genes encoding a type IV attachment pilus (Bfp) and the global virulence regulator per. Prototypic EAF plasmid pMAR7 is self-transmissible, unlike the smaller EAF plasmid pB171, which has no genes encoding conjugative functions. The tra locus, a highly conserved 33-kb segment found in pMAR7, is similar to the tra (conjugation) region of the F plasmid. ISEc13 copies flanking the pMAR7 tra region could potentially mobilize or delete the tra genes. Hybridization of 134 EPEC strains showed that a complete tra region is present only in strains of the EPEC1 clonal group. This study confirms EPEC's potential for dissemination of virulence attributes by horizontal transfer of the EAF plasmid.