The broad, long-term objectives of this proposal are to evaluate the global potential of amino acid transporterATB /ASCT2 as a selective therapeutic target in human hepatocellular carcinoma {HCC) and to elucidate thesignaling mechanisms linking it to liver cancer cell growth and survival. Currently, there are no effective therapiesfor this cancer and its incidence continues to rise in the United States. ASCT2 is expressed in all human livercancers examined to date, but is not expressed in normal human hepatocytes. Suppression of ASCT2expression by inducible antisense RNA in the SK-Hep HCC cell line leads to cell death by the intrinsic apoptoticpathway via a mechanism that is attenuated by osmotic cell swelling. Therefore, the working hypothesis is thatASCT2, via its role in substrate delivery and cellular hydration (swelling) is linked to mammalian target ofrapamycin (mTOR)- and phosphatidylinositol-3 kinase (PI3K)- dependent survival signaling pathways,respectively, that impinge upon proteins responsible for apoptotic suppression in all human liver cancer ceils.
The aims of this proposal thus arddress the universality and mechanisms linking ASCT2 to hepatoma survival:
Specific Aim 1 : To evaluate the impact of ASCT2 post-transcriptional gene silencing (PTGS) on the growth andviability of six human hepatoma cell lines, representing a broad spectrum of differentiated phenotypes.Research Plan: Several individual candidate small interfering RNA (siRNA) specific to ASCT2 will be transfectedinto all six human hepatoma cell lines, and assessed for effects on growth, viability, ASCT2 expression andcaspase activation, compared to appropriate control siRNA. Subsequently, adenoviral DMA vectors encoding forcontrol and effective siRNA's (ideally 3) will be constructed and employed in signaling experiments. Off-targetsiRNA effects in ASCT2 silenced cells will be assessed by examination of a select panel of mRNA, microarrayanalysis and by the ability of reintroduced ASCT2 to 'rescue' them. Alternative/corroborative approaches includeuse of PTGS methods such as morpholino antisense oligonucleotides or an inducible antisense RNA system.
Specific Aim 2 : To elucidate the signaling pathways linking ASCT2 silencing to hepatoma apoptosis.Research Plan: Signaling via PI3K-linked (PKB/Akt) and amino acid-dependent (mTOR) pathways as they relateto the pro-apoptotic Bcl-2 family member Bad, or other 'mitochondrial gatekeepers', will be investigated pre- andpost-ASCT2 silencing through the use of selective biochemical inhibitors and western blot analyses. Putative linksestablished through this approach will be later confirmed by the expression of dominant negative or constitutivelyactive variants of Akt or other signaling proteins. The impact of ASCT2 silencing on cell volume will be assessedby flow cytometry analysis, and reciprocally, the ability of artificial cell swelling and shrinkage to modulate thesesignaling pathways will also be investigated to better understand the mechanistic link between amino acidtransoorter function and cancer cell survival. PERFORMANCE SITE(S) (organization, city, state)Saint Louis University, St. Louis, MOKEY PERSONNEL. See instructions. Use continuation pages as needed to provide the required information in the format shown below.Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name Organization Role on ProjectBode, Barrie P. Saint Louis University Associate ProfessorFuchs, Bryan C. Saint Louis University Graduate StudentDisclosure Permission Statement Applicable to SBIR/STTR Only. See Instructions. D Y e S L'H NoPHS 398 (Rev. 05/01) Page 2 Form Page 2 Principal Investigator/Program Director (Last, first, middle): Bode Barrie P.The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANTTABLE OF CONTENTS _ M ' Page NumbersFace Page 1Description, Performance Sites, and Personnel 2-Table of Contents 3Detailed Budget for Initial Budget Period (or Modular Budget) 4Budget for Entire Proposal Period of Support (not applicable with Modular Budget)Budgets Pertaining to Consortium/Contractual Arrangements (notapplicable with Modular Budget) .Biographical Sketch-Principal Investigator/Program Director (Notto exceed four pages) 5-8Other Biographical Sketches (Notto exceed four pages for each - See instructions) 9-10Resources 11Research PlanIntroduction to Revised Application (Not toexceed3pages) 12-14Introduction to Supplemental Application (Wo/toexceedonepage) A.
Specific Aims >^ .f 1 5 B. Background andSignificance m ft. 16-18 C. Preliminary Studies/Progress Report/ . (ItemsA-D: nottoexceed25pages*) ^ 19-27 Phase I Progress Report (SBIR/STTR Phase II ONLY) I * SBIR/STTR Phase I: ItemsA-Dlimitedto 15 pages. I D. Research Design and Methods ..Jr X. 27-39 E Human Subjects 40 Protection of Human Subjects (Required if Item 4 on the Face Page is marked 'Yes') Inclusion of Women (Required if Item 4 on the Face Page is marked 'Yes') Inclusion of Minorities (Required if Item 4 on the Face Page is marked 'Yes') Inclusion of Children (Required if Item 4 on the Face Page is marked 'Yes') Data and Safety Monitoring Plan (Required if Item 4 on the Face Page is marked 'Yes'3/H/a Phase I, II, or III clinical trial isproposed) F. VertebrateAnimals 40 G. LiteratureCited 40-42 H. Consortium/Contractual Arrangements I. Letters of Support (e.g., Consultants) 43 J. Product Development Plan (SBIR/STTR Phase II and Fast-Track ONLY)Checklist 44Appendix (Five collatedsets. Nopage numbering necessary forAppendix.) Check ifAppendices NOTPERMITTED for Phase ISBIR/STTR unless specifically solicited, Appendix is IncludedNumber of publications and manuscripts accepted for publication (not to exceed10) Other items (list):PHS 398 (Rev.05/01) Form Page 3
| Fuchs, Bryan C; Finger, Richard E; Onan, Marie C et al. (2007) ASCT2 silencing regulates mammalian target-of-rapamycin growth and survival signaling in human hepatoma cells. Am J Physiol Cell Physiol 293:C55-63 |
