Document text
Principal Investigator: SHARAD KHARE
Organization: HARRY S. TRUMAN MEMORIAL VA HOSPITAL
Fiscal Year: 2024
Funding agency: Veterans Affairs
Abstract
Colorectal cancer (CRC) is the leading cause of cancer-related death in the United States. Sprouty2
(Spry2) is an endogenous suppressor of receptor tyrosine kinase (RTK)/Ras/Mitogen Activated Protein
Kinase (MAPK) signaling pathways.
The objectives of this study are:
1. To assess requirement of Spry2 in CRC mouse models.
2. To generate and analyze multi-omics data set from mouse tumors and patient organoids.
3. To assess whether differential 5mC and 5hmC hotspot loci in Spry2 may contain acquired differentially
methylated/hydroxymethlyated CpGs that can distinguish between adenomas, adenocarcinomas, and
adjacent control mucosa.
4. Furthermore, increased Spry2 mRNA expression and differential promoter methylation within regions
susceptible to 5mC changes associated with Spry2 expression may correlate with worse prognosis in
advanced rectal cancer patients undergoing preoperative chemoradiotherapy (PCRT) and surgery.
Therefore, Spry2 expression and promoter 5mC status together could serve as prognostic epimarkers
in advanced rectal cancer patients.
5. As Spry2 is regulated in CRC by DNA-methylation within the promoter and can therefore be modulated
by CRISPR-dCas9 tool to affect overall Spry2 expression and tumor growth, it could be considered as
a future treatment strategy in patients.
In previous studies, Spry2 accentuates cancer phenotype in CRC. It is hypothesized that deletion of Spry2
would suppress tumorigenesis in intestinal specific mouse models. To test this hypothesis, in Aim 1a,
Sprouty2flox/flox-Cdx2-P-Cre-ERT2, and Sprouty2flox/flox-Cdx2-P-Cre-ERT2/KrasLSL-G12D double mutant mouse
models are established and effect of Spry2 deletion on azoxymethane-induced colonic tumorigenesis is
studied. Further, Spry2 deletion in mouse models is complemented with shRNA mediated suppression of
Spry2 in Kras wild-type and KrasG12D mutant organoids established from adenomas of CRC patients (Aim
1b). Studies are extended to generate multi-omics dataset from mouse adenomas and patients’ organoids
using omics methodologies to assess signaling pathways regulated by Spry2 (Aim 1c).
Perturbing epigenetic regulation of Spry2 in patients could modify disease progression. In Aim 2a, we will
identify differentially methylated 5mC and 5hmC hotspot regions of Spry2 in CRC patients to distinguish
between adenomas, adenocarcinomas, and adjacent control mucosa. In Aim 2b, we will assess Spry2
mRNA expression and promoter methylation status in pathology collection of patients who had undergone
PCRT followed by surgery (a retrospective study) to differentiate between PCRT responders vs non-
responders by comparing disease free survival. Studies will be extended to test whether 5mC is an
epigenetic target using CRISPR-dCas9 editing in CRC. In Aim 2c, by utilizing different CRC cell lines and
immunocompromised mice, we will dissect the relationship between Spry2 methylation levels in the
promoter region with Spry2 expression and tumor growth.
The proposed study is a multi-pronged strategy to evaluate clinical significance of Spry2 in CRC.
Terms: <Adenocarcinoma><Affect><Archives><Azoxymethane><BS-seq><Binding><Biopsy><Bisulfite-based sequencing><Body Tissues><CRISPR><CRISPR/Cas system><Cancer Cause><Cancer Etiology><Cancer Genes><Cancer Patient><Cancer-Promoting Gene><Cancers><Cell Communication and Signaling><Cell Signaling><Cessation of life><Clustered Regularly Interspaced Short Palindromic Repeats><Collection><Colorectal Adenoma><Colorectal Adenomatous Polyp><Colorectal Cancer><CpG Islands><CpG-Rich Islands><DNA><DNA Methylation><Data Set><Death><Deoxyribonucleic Acid><Diazene, dimethyl-, 1-oxide><Disease Progression><Disease-Free Survival><EGF Receptor><EGFR><EGFR Blocker><EGFR Inhibitor><EGFR Tyrosine Kinase Inhibitor><EGFR-TK Inhibitor><ERBB Protein><Epidermal Growth Factor Receptor><Epidermal Growth Factor Receptor Inhibitor><Epidermal Growth Factor Receptor Kinase><Epidermal Growth Factor Receptor Protein-Tyrosine Kinase><Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor><Epidermal Growth Factor-Urogastrone Receptors><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><Epithelium><Event-Free Survival><Failure><Future><Generalized Growth><Genes><Genetic Alteration><Genetic Change><Genetic defect><Growth><HER1><High-Throughput Nucleotide Sequencing><High-Throughput Sequencing><Immunocompromised><Immunocompromised Host><Immunocompromised Patient><Immunosuppressed Host><In Vitro><Individual><Intestinal><Intestines><Intracellular Communication and Signaling><Invaded><Investigation><Investigators><KRAS(G12D)><KRASG12D><Large Bowel Adenoma><Large Bowel Adenomatous Polyp><Large Intestine Adenoma><LoxP-flanked allele><MAP kinase><Malignant Adenoma><Malignant Neoplasms><Malignant Tumor><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mediating><Mesenchymal><Messenger RNA><Methodology><Methylation><Mice><Mice Mammals><Mitogen-Activated Protein Kinases><Modeling><Molecular Interaction><Mucosa><Mucosal Tissue><Mucous Membrane><Multiomic Data><Murine><Mus><Mutation><Network Analysis><Oncogenes><Oncogenesis><Operative Procedures><Operative Surgical Procedures><Organoids><PTK Receptors><Pathology><Pathway Analysis><Patients><Phenotype><Predisposition><Prognosis><Prognostic Marker><Progress Reports><Promoter Regions><Promotor Regions><RNA Seq><RNA sequencing><RNAseq><Receptor Protein-Tyrosine Kinases><Receptor Tyrosine Kinase Gene><Rectal Cancer><Rectal Carcinoma><Regulation><Research><Research Personnel><Researchers><Retrospective Studies><Role><SCID Mice><Severe Combined Immunodeficient Mice><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Surgical><Surgical Interventions><Surgical Procedure><Susceptibility><TGF-alpha Receptor><Testing><Therapeutic Intervention><Time><Tissue Growth><Tissues><Transforming Genes><Transforming Growth Factor alpha Receptor><Transmembrane Receptor Protein Tyrosine Kinase><Tumor Suppressor Proteins><Tyrosine Kinase Linked Receptors><Tyrosine Kinase Receptors><Ulcerated Colitis><Ulcerative Colitis><United States><Update><Urogastrone Receptor><Xenograft Model><adenoma><biological signal transduction><bisulfite sequencing><bisulfite-seq><bowel><c-erbB-1><c-erbB-1 Protein><chemo-/radio-therapy><chemo-radio-therapy><chemo-radiotherapy><chemoradiation><chemoradiation therapy><chemoradiation treatment><chemoradiotherapy><clinical significance><clinically significant><colon cancer cell line><colon cancer patients><colon cancer tumorigenesis><colon tumorigenesis><colorectal cancer cell line><colorectal cancer patients><colorectal tumorigenesis><epigenetic regulation><epigenetically><erbB-1><erbB-1 Proto-Oncogene Protein><erbBl><floxed><floxed allele><genetic promoter element><genetic promoter sequence><genome mutation><immunosuppressed patient><inhibitor><intervention therapy><mRNA><mRNA Expression><malignancy><metastatic colo-rectal><metastatic colo-rectal cancer><metastatic colo-rectal carcinoma><metastatic colon cancer><metastatic colorectal><metastatic colorectal cancer><metastatic colorectal carcinoma><mouse model><multiple omic data><murine model><mutant><mutant mouse model><neoplasm/cancer><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><novel><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><ontogeny><postnatal><prognostic><prognostic biomarker><promoter><promoter sequence><promotor><proto-oncogene protein c-erbB-1><pyrosequencing><radio-chemo-therapy><radio-chemotherapy><radiochemotherapy><resistance mechanism><resistant mechanism><responders and non-responders><responders from non-responders><responders or non-responders><responders versus non-responders><responders vs non-responders><responders/nonresponders><shRNA><short hairpin RNA><small hairpin RNA><social role><surgery><targeted drug therapy><targeted drug treatments><targeted therapeutic><targeted therapeutic agents><targeted therapy><targeted treatment><tool><transcriptome sequencing><transcriptomic sequencing><treatment strategy><tumor><tumor growth><tumor suppressor><tumorigenesis><xenograft transplant model><xenotransplant model>