The combination of an mTORc1/TORc2 inhibitor with lapatinib is synergistic in bladder cancer in vitro.
OBJECTIVE
To examine the ability of dual mTORc1/c2 inhibitors in conjunction with lapatinib to function in a synergistic manner to inhibit cell proliferation and anchorage-independent growth in bladder cancer cell lines.


METHODS
We examined patient tumor samples for overexpression of pS6, p4EBP1, pAkt, and phosphorylated epidermal growth factor receptor (pEGFR) using a tissue microarray containing 84 cases. Three bladder cancer cell lines, T24, HT1376, and UM-UC-3, were analyzed for cell proliferation after treatment with mTORc1/c2 inhibitors OSI-027 or PP242. Western blots were used to verify that the drugs were inhibiting phosphorylation of target proteins within the mTOR pathway, and they were compared with rapamycin inhibition. We also analyzed cell proliferation and anchorage-independent growth after treatment with OSI-027 and lapatinib in combination. PARP cleavage and autophagic flux were measured by examining levels of LC3B and p62 by western blotting.


RESULTS
Tumor samples show increased expression of pEGFR (38% vs. 8%) and HER2 (38% vs. 4%) and decreased expression of pAkt S473 (7.5% vs. 29%) and pAkt T308 (50% vs. 84%) relative to normal tissue. Significant differences between normal and tumor samples for staining with pEGFR (P = 0.0188), HER 2 (P = 0.0017), pATK S473 (P = 0.0128), and pAkt T308 (P = 0.0015) is observed. Expression of proteins within the EGFR/HER2 pathway or within the mTOR pathway is correlated. No correlation was found between staining and tumor stage. OSI-027 and PP242 diminish cell proliferation in all 3 cell lines with IC50 values ranging from 0.63 to 17.95µM. Both drugs inhibit phosphorylation of both mTORc1 and mTORc2 pathway components. OSI-027 and lapatinib inhibit cell proliferation and anchorage-independent growth in a synergistic manner. One cell line exhibited apoptosis in response to combination drug treatment, whereas the other 2 cell lines have increased levels of autophagy indicative of resistance to apoptosis.


CONCLUSIONS
The combination of OSI-027 and lapatinib results in antitumor synergy and further exploration of this combination should be undertaken.
Division of Urology, Department of Surgery, College of Medicine-Jacksonville, University of Florida, Jacksonville, FL. Electronic address: Marie.becker@jax.ufl.edu.
Marie N
MN
Becker
Department of Laboratory Medicine and Pathology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic, Jacksonville, FL.
Kevin J
KJ
Wu
Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic, Jacksonville, FL.
Laura A
LA
Marlow
Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic, Jacksonville, FL.
Pamela A
PA
Kreinest
Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic, Jacksonville, FL.
Christina A
CA
Vonroemeling
Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic, Jacksonville, FL.
John A
JA
Copland
Division of Urology, Department of Surgery, College of Medicine-Jacksonville, University of Florida, Jacksonville, FL.
Christopher R
CR
Williams
D000368:Aged; D000970:Antineoplastic Agents / Q000008:administration & dosage*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004357:Drug Synergism; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000008:administration & dosage*; D007150:Immunohistochemistry; D007211:Indoles / Q000008:administration & dosage; D000077341:Lapatinib; D008297:Male; D011687:Purines / Q000008:administration & dosage; D011799:Quinazolines / Q000008:administration & dosage*; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D046888:Tissue Array Analysis; D014227:Triazines / Q000008:administration & dosage*; D001749:Urinary Bladder Neoplasms / Q000473:pathology*
D000970:Antineoplastic Agents; D007093:Imidazoles; D007211:Indoles; C568605:OSI 027; D011687:Purines; D011799:Quinazolines; D014227:Triazines; D000077341:Lapatinib; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; C572919:PP242
Antitumor synergy; Bladder cancer; EGFR; Lapatinib; mTOR