Portfolio of Past Work Nish Patel, Ph.D.
Postdoctoral Research Harvard Medical School Sept. 2006 – Dec. 2009
Multi-Drug Resistance <ul><li>~ 40% of human tumors develop resistance to chemotherapeutic drugs </li></ul>
<ul><li>Paclitaxel® (Taxol) </li></ul><ul><li>a frontline chemotherapeutic agent  </li></ul><ul><li>binds to   -tubulin p...
Rationale: Increased understanding of the molecules associated with drug resistance would undoubtedly be useful for the di...
<ul><li>Prohibitin1 </li></ul><ul><li>molecular mass of 30-32 kDa </li></ul><ul><li>Prohibitin1 forms a high molecular wei...
Prohibitin expression is elevated in Paclitaxel resistant cell lines Paclitaxel Resistance Paclitaxel Resistance -  +  -  ...
Prohibitin1 localizes to the mitochondria in both drug-sensitive and resistant cell lines Paclitaxel-sensitive Paclitaxel-...
Prohibitin1 is localized to the cell surface in drug resistant cell lines Phb1 Antibody Rhodamine-PHB1-binding peptide Pac...
Prohibitin1-binding peptide preferentially localizes to Paclitaxel-resistant xenografts after  intravenous injection Rhoda...
Effect of  Prohibitin1 siRNA on Taxol Sensitivity of  Paclitaxel-resistant Cells
The generation of Paclitaxel-resistant cell lines with stable knockdown of Prohibitin (PHB1) or GST 
Paclitaxel sensitivity in stable cell line expressing PHB1 or GST   shRNA
Paclitaxel Sensitivity of A549TR xenografts expressing control shRNA
Paclitaxel Sensitivity of A549TR xenografts expressing PHB1 shRNA
Paclitaxel Sensitivity of A549TR xenografts expressing GST   shRNA
Paclitaxel Sensitivity of A549TR xenografts expressing control, PHB1 or GST   shRNA
Paclitaxel Sensitivity of A549TR xenografts expressing control, PHB1 or GST   shRNA
EMERGING HYPOTHESIS: The selective spatial localization of Prohibitin1 may be necessary for  the onset and/or maintenance ...
SIGNIFICANCE: Prohibitin1 on the surface of tumor cells may represent a novel marker  for the detection of taxane resistan...
Ph. D. Candidate Research University of Toronto Jan. 2001 – Mar. 2006
Insulin Stimulates Glucose Uptake Pancreas GLUT4 is the major insulin-responsive  glucose transporter
Several stimuli stimulate GLUT4 translocation Stress
<ul><li>How is signal specificity achieved? </li></ul><ul><li>Activation of unique signalling molecules </li></ul><ul><li>...
GLUT4 Glucose
Insulin induces   GFP-actin   remodelling and membrane ruffling in L6 skeletal muscle cells Confocal Fluorescence Microsco...
HYPOTHESIS:   Insulin-induced actin remodelling is needed for signalling molecules to meet with GLUT4 in time and space
<ul><li>OBJECTIVE:   </li></ul><ul><li>To determine the spatial localization of insulin signalling molecules  vis a vis  t...
Insulin-mediated cellular redistribution of   GFP-PH-GRP1   reflects its PI-3,4,5-P 3  binding capacity Patel et al.,  Mol...
Insulin-induced actin remodelling promotes the spatial localization of specific signalling molecules within the actin mesh
<ul><li>OBJECTIVE:   </li></ul><ul><li>To determine the spatial localization of insulin signalling molecules  vis a vis  t...
GLUT4 (10 nm; arrowhead) and p110   (5 nm; arrow) colocalize within insulin-induced actin structures Basal Insulin
<ul><li>OBJECTIVE:   </li></ul><ul><li>To determine the spatial localization of insulin signalling molecules  vis a vis  t...
<ul><li>member of  A ctin depolymerizing factor/ C ofilin (AC) family </li></ul><ul><li>21 kDa </li></ul><ul><li>ubiquitou...
Cofilin1 siRNA disrupts insulin-induced actin remodelling
Cofilin1 siRNA inhibits insulin-induced GLUT4myc translocation * p  < 0.005, n = 6
EMERGING HYPOTHESIS: The selective spatial localization of insulin signalling molecules  within the actin mesh may ultimat...
SIGNIFICANCE: A defect in insulin signalling leading to actin remodelling may  contribute to insulin resistance in the ske...
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Portfolio of Past Research

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A sampling of my past research findings as a Research Fellow at Harvard Medical School and as a Ph.D. candidate at the University of Toronto.

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  • ~ 40% of human tumors develop resistance to chemotherapeutic drugs While it is thought that the majority of cancers arise from a single precursor cell, it would be an  error to view a tumor as consisting of a collection of genetically identical cells. One of the hallmarks of cancer is an increase in genetic instability and mutation rates. These changes mean that dividing cancer cells acquire genetic changes at a high rate. Practically, this means that the cells in a tumor, while similar, are NOT identical. When exposed to a cancer drug, those cells that are sensitive to the effects of the drug are killed. Those that are resistant will survive and multiply. The result is the re-growth of a tumor that is not sensitive to the original drug. Surprisingly, cells simultaneously acquire resistance to many chemically unrelated compounds to which the cell has never been exposed to. This phenomenon is referred to as multi drug resistance.
  • In order to validate the mass-spec data, cytoplasmic and microsomal fractions were isolated and probed for Prohibitin and GSTpi by western blot analysis.
  • A key regulator of actin dynamics we decided to focus on is cofilin. Cofilin is a highly expressed in various cell types and it functions as an actin depolymerizer. Upon binding to actin, cofilin induces a twist in the filament, and this conformational change weakens contacts between actin subunits, enhancing the depolymerization of actin. In the inactive state, cofilin is phosphorylated at Ser-3 (as shown here) and this prevents the depolymerization of F-actin. In contrast, active cofilin lacks phosphorylation at Ser-3, thus allowing for actin depolyermization.
  • Portfolio of Past Research

    1. 1. Portfolio of Past Work Nish Patel, Ph.D.
    2. 2. Postdoctoral Research Harvard Medical School Sept. 2006 – Dec. 2009
    3. 3. Multi-Drug Resistance <ul><li>~ 40% of human tumors develop resistance to chemotherapeutic drugs </li></ul>
    4. 4. <ul><li>Paclitaxel® (Taxol) </li></ul><ul><li>a frontline chemotherapeutic agent </li></ul><ul><li>binds to  -tubulin preventing microtubule dynamics </li></ul><ul><li>FDA approved in 1992 for treatment of metastatic breast cancer </li></ul><ul><li>Clinically approved for the treatment of breast and ovarian cancer, in clinical trial for prostate, lung and several other cancers </li></ul><ul><li>Major limitation is its susceptible to several mechanisms to drug resistance </li></ul><ul><ul><li>Expression of the MDR phenotype </li></ul></ul><ul><ul><li>Alterations of the microtubule system </li></ul></ul>
    5. 5. Rationale: Increased understanding of the molecules associated with drug resistance would undoubtedly be useful for the discovery of new targets for the detection, delay or reversal of resistance. Objective: Identify proteins associated with Paclitaxel ® resistance and to examine their potential use as targets for modulating the resistant phenotype
    6. 6. <ul><li>Prohibitin1 </li></ul><ul><li>molecular mass of 30-32 kDa </li></ul><ul><li>Prohibitin1 forms a high molecular weight complex with Prohibitin2 </li></ul><ul><li>primarily localized to the inner mitochondrial membrane but also found on the cell surface </li></ul><ul><li>Best characterized function of prohibitin is as a chaperone </li></ul><ul><li>Interacts with E2F, p53, and pRb in the nucleus </li></ul>Vol. 11 No. 4 April 2005 pp192-197
    7. 7. Prohibitin expression is elevated in Paclitaxel resistant cell lines Paclitaxel Resistance Paclitaxel Resistance - + - + - + - + Cell line 1 Cell line 2 Cell line 1 Cell line 2
    8. 8. Prohibitin1 localizes to the mitochondria in both drug-sensitive and resistant cell lines Paclitaxel-sensitive Paclitaxel-resistant
    9. 9. Prohibitin1 is localized to the cell surface in drug resistant cell lines Phb1 Antibody Rhodamine-PHB1-binding peptide Paclitaxel-sensitive Paclitaxel-resistant Paclitaxel-sensitive Paclitaxel-resistant
    10. 10. Prohibitin1-binding peptide preferentially localizes to Paclitaxel-resistant xenografts after intravenous injection Rhodamine-PHB1-binding peptide Rhodamine-PHB1-binding peptide DAPI DAPI Paclitaxel-sensitive Paclitaxel-resistant
    11. 11. Effect of Prohibitin1 siRNA on Taxol Sensitivity of Paclitaxel-resistant Cells
    12. 12. The generation of Paclitaxel-resistant cell lines with stable knockdown of Prohibitin (PHB1) or GST 
    13. 13. Paclitaxel sensitivity in stable cell line expressing PHB1 or GST  shRNA
    14. 14. Paclitaxel Sensitivity of A549TR xenografts expressing control shRNA
    15. 15. Paclitaxel Sensitivity of A549TR xenografts expressing PHB1 shRNA
    16. 16. Paclitaxel Sensitivity of A549TR xenografts expressing GST  shRNA
    17. 17. Paclitaxel Sensitivity of A549TR xenografts expressing control, PHB1 or GST  shRNA
    18. 18. Paclitaxel Sensitivity of A549TR xenografts expressing control, PHB1 or GST  shRNA
    19. 19. EMERGING HYPOTHESIS: The selective spatial localization of Prohibitin1 may be necessary for the onset and/or maintenance for paclitaxel-resistance in human cancers.
    20. 20. SIGNIFICANCE: Prohibitin1 on the surface of tumor cells may represent a novel marker for the detection of taxane resistance in cancer patients either before or during the course of chemotherapy. It may also act as a novel target for therapeutic strategies for the treatment of drug-resistant tumors.
    21. 21. Ph. D. Candidate Research University of Toronto Jan. 2001 – Mar. 2006
    22. 22. Insulin Stimulates Glucose Uptake Pancreas GLUT4 is the major insulin-responsive glucose transporter
    23. 23. Several stimuli stimulate GLUT4 translocation Stress
    24. 24. <ul><li>How is signal specificity achieved? </li></ul><ul><li>Activation of unique signalling molecules </li></ul><ul><li>Signal amplitude and time course </li></ul><ul><li>Unique subcellular loci </li></ul>
    25. 25. GLUT4 Glucose
    26. 26. Insulin induces GFP-actin remodelling and membrane ruffling in L6 skeletal muscle cells Confocal Fluorescence Microscopy Scanning Electron Microscopy
    27. 27. HYPOTHESIS: Insulin-induced actin remodelling is needed for signalling molecules to meet with GLUT4 in time and space
    28. 28. <ul><li>OBJECTIVE: </li></ul><ul><li>To determine the spatial localization of insulin signalling molecules vis a vis the remodelled actin cytoskeleton </li></ul><ul><li>Determine the ultrastructure of the actin cytoskeleton by electron microscopy </li></ul><ul><li>Identify signalling molecules that regulate in insulin-stimulated actin remodelling and GLUT4 translocation </li></ul>
    29. 29. Insulin-mediated cellular redistribution of GFP-PH-GRP1 reflects its PI-3,4,5-P 3 binding capacity Patel et al., Mol. Cell Biol. , 2003 Insulin
    30. 30. Insulin-induced actin remodelling promotes the spatial localization of specific signalling molecules within the actin mesh
    31. 31. <ul><li>OBJECTIVE: </li></ul><ul><li>To determine the spatial localization of insulin signalling molecules vis a vis the remodelled actin cytoskeleton </li></ul><ul><li>Determine the ultrastructure of the actin cytoskeleton by electron microscopy </li></ul><ul><li>Identify signalling molecules that regulate insulin-stimulated actin remodelling and GLUT4 translocation </li></ul>
    32. 32. GLUT4 (10 nm; arrowhead) and p110  (5 nm; arrow) colocalize within insulin-induced actin structures Basal Insulin
    33. 33. <ul><li>OBJECTIVE: </li></ul><ul><li>To determine the spatial localization of insulin signalling molecules vis a vis the remodelled actin cytoskeleton </li></ul><ul><li>Determine the ultrastructure of the actin cytoskeleton by electron microscopy </li></ul><ul><li>Identify the signalling molecules that regulate insulin-stimulated actin remodelling and GLUT4 translocation </li></ul>
    34. 34. <ul><li>member of A ctin depolymerizing factor/ C ofilin (AC) family </li></ul><ul><li>21 kDa </li></ul><ul><li>ubiquitously expressed in varying cell types </li></ul><ul><li>binds F-actin and G-actin </li></ul>Cofilin: a regulator of actin dynamics <ul><li>Active State = unphosphorylated at Ser-3 </li></ul>Hypothesis: Cofilin is necessary for insulin-stimulated actin remodelling and GLUT4 translocation to the cell surface
    35. 35. Cofilin1 siRNA disrupts insulin-induced actin remodelling
    36. 36. Cofilin1 siRNA inhibits insulin-induced GLUT4myc translocation * p < 0.005, n = 6
    37. 37. EMERGING HYPOTHESIS: The selective spatial localization of insulin signalling molecules within the actin mesh may ultimately lead to GLUT4 arrival at the plasma membrane.
    38. 38. SIGNIFICANCE: A defect in insulin signalling leading to actin remodelling may contribute to insulin resistance in the skeletal muscle and to the onset of type 2 diabetes.

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