Friendly fire Snook, Scott A; Snook, Scott A
2011., 20110919, 2011, 2000-01-01
eBook
On April 14, 1994, two U.S. Air Force F-15 fighters accidentally shot down two U.S. Army Black Hawk Helicopters over Northern Iraq, killing all twenty-six peacekeepers onboard. In response to this ...disaster the complete array of military and civilian investigative and judicial procedures ran their course. After almost two years of investigation with virtually unlimited resources, no culprit emerged, no bad guy showed himself, no smoking gun was found. This book attempts to make sense of this tragedy--a tragedy that on its surface makes no sense at all.
Visceral pain (VP) is a global problem with complex etiologies and limited therapeutic options. Guanylyl cyclase C (GUCY2C), an intestinal receptor producing cyclic GMP(cGMP), which regulates luminal ...fluid secretion, has emerged as a therapeutic target for VP. Indeed, FDA-approved GUCY2C agonists ameliorate VP in patients with chronic constipation syndromes, although analgesic mechanisms remain obscure. Here, we revealed that intestinal GUCY2C was selectively enriched in neuropod cells, a type of enteroendocrine cell that synapses with submucosal neurons in mice and humans. GUCY2Chi neuropod cells associated with cocultured dorsal root ganglia neurons and induced hyperexcitability, reducing the rheobase and increasing the resulting number of evoked action potentials. Conversely, the GUCY2C agonist linaclotide eliminated neuronal hyperexcitability produced by GUCY2C-sufficient - but not GUCY2C-deficient - neuropod cells, an effect independent of bulk epithelial cells or extracellular cGMP. Genetic elimination of intestinal GUCY2C amplified nociceptive signaling in VP that was comparable with chemically induced VP but refractory to linaclotide. Importantly, eliminating GUCY2C selectively in neuropod cells also increased nociceptive signaling and VP that was refractory to linaclotide. In the context of loss of GUCY2C hormones in patients with VP, these observations suggest a specific role for neuropod GUCY2C signaling in the pathophysiology and treatment of these pain syndromes.
: Colorectal cancer (CRC) is one of the most common forms of cancer worldwide and is the second leading cause of cancer-related death in the United States. Despite advances in early detection, ~25% ...of patients are late stage, and treated patients have <12% chance of survival after five years. Tumor relapse and metastasis are the main causes of patient death. Cancer stem cells (CSCs) are a rare population of cancer cells characterized by properties of self-renewal, chemo- and radio-resistance, tumorigenicity, and high plasticity. These qualities make CSCs particularly important for metastasic seeding, DNA-damage resistance, and tumor repopulating.
: The following review article focuses on the role of CRC-SCs in tumor initiation, metastasis, drug resistance, and tumor relapse, as well as on potential therapeutic options for targeting CSCs.
: Current studies are underway to better isolate and discriminate CSCs from normal stem cells and to produce CSC-targeted therapeutics. The intestinal receptor, guanylate cyclase C (GUCY2C) could potentially provide a unique therapeutic target for both non-stem cells and CSCs alike in colorectal cancer through immunotherapies. Indeed, immunotherapies targeting CSCs have the potential to break the treatment-recurrence cycle in the management of advanced malignancies.
Neurological disorders are the leading cause of cognitive and physical disability worldwide, affecting 15% of the global population. Due to the demographics of aging, the prevalence of neurological ...disorders, including neurodegenerative diseases, will double over the next two decades. Unfortunately, while available therapies provide symptomatic relief for cognitive and motor impairment, there is an urgent unmet need to develop disease-modifying therapies that slow the rate of pathological progression. In that context, biomarkers could identify at-risk and prodromal patients, monitor disease progression, track responses to therapy, and parse the causality of molecular events to identify novel targets for further clinical investigation. Thus, identifying biomarkers that discriminate between diseases and reflect specific stages of pathology would catalyze the discovery and development of therapeutic targets. This review will describe the prevalence, known mechanisms, ongoing or recently concluded therapeutic clinical trials, and biomarkers of three of the most prevalent neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD).
Obesity is a well-known risk factor for colorectal cancer but precisely how it influences risks of malignancy remains unclear. During colon cancer development in humans or animals, attenuation of the ...colonic cell surface receptor guanylyl cyclase C (GUCY2C) that occurs due to loss of its paracrine hormone ligand guanylin contributes universally to malignant progression. In this study, we explored a link between obesity and GUCY2C silencing in colorectal cancer. Using genetically engineered mice on different diets, we found that diet-induced obesity caused a loss of guanylin expression in the colon with subsequent GUCY2C silencing, epithelial dysfunction, and tumorigenesis. Mechanistic investigations revealed that obesity reversibly silenced guanylin expression through calorie-dependent induction of endoplasmic reticulum stress and the unfolded protein response in intestinal epithelial cells. In transgenic mice, enforcing specific expression of guanylin in intestinal epithelial cells restored GUCY2C signaling, eliminating intestinal tumors associated with a high calorie diet. Our findings show how caloric suppression of the guanylin-GUCY2C signaling axis links obesity to negation of a universal tumor suppressor pathway in colorectal cancer, suggesting an opportunity to prevent colorectal cancer in obese patients through hormone replacement with the FDA-approved oral GUCY2C ligand linaclotide.
The Gram-positive bacterium
(Lm) is an emerging platform for cancer immunotherapy. To date, over 30 clinical trials have been initiated testing Lm cancer vaccines across a wide variety of cancers, ...including lung, cervical, colorectal, and pancreatic. Here, we assessed the immunogenicity of an Lm vaccine against the colorectal tumor antigen GUCY2C (Lm-GUCY2C). Surprisingly, Lm-GUCY2C vaccination did not prime naïve GUCY2C-specific CD8
T-cell responses towards the dominant H-2K
-restricted epitope, GUCY2C
. However, Lm-GUCY2C produced robust CD8
T-cell responses towards Lm-derived peptides suggesting that GUCY2C
peptide may be subdominant to Lm-derived peptides. Indeed, incorporating immunogenic Lm peptides into an adenovirus-based GUCY2C vaccine previously shown to induce robust GUCY2C
immunity completely suppressed GUCY2C
responses. Comparison of immunogenic Lm-derived peptides to GUCY2C
revealed that Lm-derived peptides form highly stable peptide-MHC complexes with H-2K
compared to GUCY2C
peptide. Moreover, amino acid substitution at a critical anchoring residue for H-2K
binding, producing GUCY2C
, significantly improved stability with H-2K
and rescued GUCY2C
immunogenicity in the context of Lm vaccination. Collectively, these studies suggest that Lm antigens may compete with and suppress the immunogenicity of target vaccine antigens and that use of altered peptide ligands with enhanced peptide-MHC stability may be necessary to elicit robust immune responses. These studies suggest that optimizing target antigen competitiveness with Lm antigens or alternative immunization regimen strategies, such as prime-boost, may be required to maximize the clinical utility of Lm-based vaccines.
There is a geographic inequality in the incidence of colorectal cancer, lowest in developing countries, and greatest in developed countries. This disparity suggests an environmental contribution to ...cancer resistance in endemic populations. Enterotoxigenic bacteria associated with diarrheal disease are prevalent in developing countries, including enterotoxigenic
(
) producing heat-stable enterotoxins (STs). STs are peptides that are structurally homologous to paracrine hormones that regulate the intestinal guanylyl cyclase C (GUCY2C) receptor. Beyond secretion, GUCY2C is a tumor suppressor universally silenced by loss of expression of its paracrine hormone during carcinogenesis. Thus, the geographic imbalance in colorectal cancer, in part, may reflect chronic exposure to ST-producing organisms that restore GUCY2C signaling silenced by hormone loss during transformation. Here, mice colonized for 18 weeks with control
or those engineered
secrete ST exhibited normal growth, with comparable weight gain and normal stool water content, without evidence of secretory diarrhea. Enterotoxin-producing, but not control,
, generated ST that activated colonic GUCY2C signaling, cyclic guanosine monophosphate (cGMP) production, and cGMP-dependent protein phosphorylation in colonized mice. Moreover, mice colonized with ST-producing
exhibited a 50% reduction in carcinogen-induced colorectal tumor burden. Thus, chronic colonization with
producing ST could contribute to endemic cancer resistance in developing countries, reinforcing a novel paradigm of colorectal cancer chemoprevention with oral GUCY2C-targeted agents.
Background
Colorectal cancer most commonly arises from mutations in the tumor suppressor APC, or its downstream degradation target β‐catenin, initiating an oncogenic program of ...β‐catenin/TCF‐dependent nuclear transcription. Mechanisms coupling these mutations to tumorigenesis continue to be refined. The tumor suppressor axis regulated by the intestinal receptor guanylyl cyclase C (GUCY2C) and its paracrine ligands, guanylin and uroguanylin, contributes to intestinal homeostasis by regulating the proliferation, migration, and differentiation programs that maintain intestinal epithelial architecture. Furthermore, GUCY2C agonists oppose transformation in multiple mouse models of intestinal tumorigenesis. This signaling axis is among the earliest pathways silenced in tumorigenesis, reflecting an evolutionarily conserved pattern of GUCY2C retention, but ligand loss, in tumors. These observations suggest a pathophysiological model in which transformation orphans the receptor, silencing its signaling and lifting a block on tumorigenesis. Here, we examined the hypothesis that GUCY2C ligand loss arises from transcriptional silencing by β‐catenin/TCF.
Methods
We mapped the β‐catenin/TCF‐transcriptional program by RNA‐seq in four human colon cancer cell models. We then performed a comparative analysis of orthogonal approaches, including luciferase reporters, ChIP‐seq, CRISPR Cas9 knockout, and CRISPR epigenome editing to identify gene enhancers mediating GUCY2C ligand loss.
Results
RNA‐seq revealed a core β‐catenin/TCF‐transcriptional program of 1,289 genes, of which guanylin and uroguanylin were the seventh and twelfth most sensitive transcripts, reflecting transcriptional silencing. ChIP‐seq analyses of colon tissue revealed an evolutionarily conserved genomic locus encompassing the GUCY2C ligand genes, containing multiple regions of DNase hypersensitivity and H3K27ac enrichment, hallmarks of genetic enhancer loci. These markers were observed in normal colon, but not cancer tissue, consistent with enhancer inactivation and transcriptional silencing during transformation. ChIP‐seq in a colon cancer cell line revealed RNA Polymerase II and H3K27ac recruitment to the regions identified in human tissue, reflecting poised transcriptional machinery. Incorporation of the putative enhancer DNA into luciferase reporter constructs revealed a 2,683 bp locus control region (LCR) responsible for coordinated β‐catenin/TCF‐sensitive control of both GUCY2C ligand promoters. Both CRISPR‐Cas9 deletion of the LCR and epigenetic inactivation with a dCas9.KRAB repressor construct abolished β‐catenin/TCF‐sensitivity of GUCY2C ligand expression. Finally, LCR activation with a dCas9.VP64 transcriptional activator construct reconstituted GUCY2C ligand expression, overcoming gene silencing by β‐catenin/TCF.
Conclusions
This study reveals DNA elements regulating co‐repression of GUCY2C ligand transcription by β‐catenin/TCF, reflecting a novel step in tumorigenesis, and offers unique strategies to re‐establish hormone expression to oppose transformation.
One major hurdle to the success of adoptive T-cell therapy is the identification of antigens that permit effective targeting of tumors in the absence of toxicities to essential organs. Previous work ...has demonstrated that T cells engineered to express chimeric antigen receptors (CAR-T cells) targeting the murine homolog of the colorectal cancer antigen GUCY2C treat established colorectal cancer metastases, without toxicity to the normal GUCY2C-expressing intestinal epithelium, reflecting structural compartmentalization of endogenous GUCY2C to apical membranes comprising the intestinal lumen. Here, we examined the utility of a human-specific, GUCY2C-directed single-chain variable fragment as the basis for a CAR construct targeting human GUCY2C-expressing metastases. Human GUCY2C-targeted murine CAR-T cells promoted antigen-dependent T-cell activation quantified by activation marker upregulation, cytokine production, and killing of GUCY2C-expressing, but not GUCY2C-deficient, cancer cells
GUCY2C CAR-T cells provided long-term protection against lung metastases of murine colorectal cancer cells engineered to express human GUCY2C in a syngeneic mouse model. GUCY2C murine CAR-T cells recognized and killed human colorectal cancer cells endogenously expressing GUCY2C, providing durable survival in a human xenograft model in immunodeficient mice. Thus, we have identified a human GUCY2C-specific CAR-T cell therapy approach that may be developed for the treatment of GUCY2C-expressing metastatic colorectal cancer.
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