p53 promotes revival stem cells in the regenerating intestine after severe radiation injury.

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced acute GI syndrome. Through single-cell RNA-sequencing of the irradiated mouse small intestine, we find that p53 target genes are specifically enriched in regenerating epithelial cells that undergo fetal-like reversion, including revival stem cells (revSCs) that promote animal survival after severe damage of the GI tract. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce fetal-like revSCs. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells and is controlled by an Mdm2-mediated negative feedback loop. Together, our findings reveal that p53 suppresses severe radiation-induced GI injury by promoting fetal-like reprogramming of irradiated intestinal epithelial cells.

Histological assessment of intestinal injury by ionizing radiation.

Given the potential risk of radiological terrorism and disasters, it is essential to develop plans to prepare for such events. In these hazardous scenarios, radiation-induced gastrointestinal (GI) syndrome is one of the many manifestations that may happen after the organism is exposed to a lethal dose of ionizing radiation. Therefore, it is critical to better understand how the intestinal tissues initiate and orchestrate regeneration following severe radiation injury. In this chapter, we aimed to provide several key considerations for researchers who utilize histological assessment to study radiation-induced intestinal injury. Rigor and reproducibility are critical in experimental design and can be achieved by maintaining proper radiation administration, maintaining consistency in sample collection, and selecting and using appropriate controls. We also provided technical details of histological preparation of the intestines with tips on dissecting, cleaning, fixing, and preserving. Step-by-step descriptions of both bundling and Swiss rolling are provided with discussion on how to choose between the two approaches. In the following section, we detailed several histological assessment methods and then provided suggestions on how to use histological assessment to study cellular dynamics in the small intestines. Finally, we touched on some non-histological assessments. We hope that the information provided in this chapter will contribute to the research society of radiation-induced intestinal injury with an ultimate goal of promoting the development of radiation countermeasures against the GI acute radiation syndrome.

p53 promotes revival stem cells in the regenerating intestine after severe radiation injury.

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced GI injury. Through single-cell RNA-sequencing of the irradiated mouse intestine, we find that p53 target genes are specifically enriched in stem cells of the regenerating epithelium, including revival stem cells that promote animal survival after GI damage. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce revival stem cells. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells that is controlled by an Mdm2-mediated negative feedback loop. These results suggest that p53 suppresses severe radiation-indued GI injury by promoting intestinal epithelial cell reprogramming. One-Sentence Summary: After severe radiation injury to the intestine, transient p53 activity induces revival stem cells to promote regeneration.

Non-canonical role of Hippo tumor suppressor serine/threonine kinase 3 STK3 in prostate cancer.

Serine/threonine kinase 3 (STK3) is an essential member of the highly conserved Hippo tumor suppressor pathway that regulates Yes-associated protein 1 (YAP1) and TAZ. STK3 and its paralog STK4 initiate a phosphorylation cascade that regulates YAP1/TAZ inhibition and degradation, which is important for regulated cell growth and organ size. Deregulation of this pathway leads to hyperactivation of YAP1 in various cancers. Counter to the canonical tumor suppression role of STK3, we report that in the context of prostate cancer (PC), STK3 has a pro-tumorigenic role. Our investigation started with the observation that STK3, but not STK4, is frequently amplified in PC. Additionally, high STK3 expression is associated with decreased overall survival and positively correlates with androgen receptor (AR) activity in metastatic castrate-resistant PC. XMU-MP-1, an STK3/4 inhibitor, slowed cell proliferation, spheroid growth, and Matrigel invasion in multiple models. Genetic depletion of STK3 decreased proliferation in several PC cell lines. In a syngeneic allograft model, STK3 loss slowed tumor growth kinetics in vivo, and biochemical analysis suggests a mitotic growth arrest phenotype. To further probe the role of STK3 in PC, we identified and validated a new set of selective STK3 inhibitors, with enhanced kinase selectivity relative to XMU-MP-1, that inhibited tumor spheroid growth and invasion. Consistent with the canonical role, inhibition of STK3 induced cardiomyocyte growth and had chemoprotective effects. Our results indicate that STK3 has a non-canonical role in PC progression and that inhibition of STK3 may have a therapeutic potential for PC that merits further investigation.

NUAK family kinase 2 is a novel therapeutic target for prostate cancer.

Current advancements in prostate cancer (PC) therapies have been successful in slowing PC progression and increasing life expectancy; however, there is still no curative treatment for advanced metastatic castration resistant PC (mCRPC). Most treatment options target the androgen receptor, to which many PCs eventually develop resistance. Thus, there is a dire need to identify and validate new molecular targets for treating PC. We found NUAK family kinase 2 (NUAK2) expression is elevated in PC and mCRPC versus normal tissue, and expression correlates with an increased risk of metastasis. Given this observation and because NUAK2, as a kinase, is actionable, we evaluated the potential of NUAK2 as a molecular target for PC. NUAK2 is a stress response kinase that also plays a role in activation of the YAP cotranscriptional oncogene. Combining pharmacological and genetic methods for modulating NUAK2, we found that targeting NUAK2 in vitro leads to reduction in proliferation, three-dimensional tumor spheroid growth, and matrigel invasion of PC cells. Differential gene expression analysis of PC cells treated NUAK2 small molecule inhibitor HTH-02-006 demonstrated that NUAK2 inhibition results in downregulation of E2F, EMT, and MYC hallmark gene sets after NUAK2 inhibition. In a syngeneic allograft model and in radical prostatectomy patient derived explants, NUAK2 inhibition slowed tumor growth and proliferation rates. Mechanistically, HTH-02-006 treatment led to inactivation of YAP and the downregulation of NUAK2 and MYC protein levels. Our results suggest that NUAK2 represents a novel actionable molecular target for PC that warrants further exploration.

27-Hydroxycholesterol Impairs Plasma Membrane Lipid Raft Signaling as Evidenced by Inhibition of IL6-JAK-STAT3 Signaling in Prostate Cancer Cells.

We recently reported that restoring the CYP27A1-27hydroxycholesterol axis had antitumor properties. Thus, we sought to determine the mechanism by which 27HC exerts its anti-prostate cancer effects. As cholesterol is a major component of membrane microdomains known as lipid rafts, which localize receptors and facilitate cellular signaling, we hypothesized 27HC would impair lipid rafts, using the IL6-JAK-STAT3 axis as a model given its prominent role in prostate cancer. As revealed by single molecule imaging of DU145 prostate cancer cells, 27HC treatment significantly reduced detected cholesterol density on the plasma membranes. Further, 27HC treatment of constitutively active STAT3 DU145 prostate cancer cells reduced STAT3 activation and slowed tumor growth in vitro and in vivo. 27HC also blocked IL6-mediated STAT3 phosphorylation in nonconstitutively active STAT3 cells. Mechanistically, 27HC reduced STAT3 homodimerization, nuclear translocation, and decreased STAT3 DNA occupancy at target gene promoters. Combined treatment with 27HC and STAT3 targeting molecules had additive and synergistic effects on proliferation and migration, respectively. Hallmark IL6-JAK-STAT gene signatures positively correlated with CYP27A1 gene expression in a large set of human metastatic castrate-resistant prostate cancers and in an aggressive prostate cancer subtype. This suggests STAT3 activation may be a resistance mechanism for aggressive prostate cancers that retain CYP27A1 expression. In summary, our study establishes a key mechanism by which 27HC inhibits prostate cancer by disrupting lipid rafts and blocking STAT3 activation. IMPLICATIONS: Collectively, these data show that modulation of intracellular cholesterol by 27HC can inhibit IL6-JAK-STAT signaling and may synergize with STAT3-targeted compounds.