AWARD NUMBER: DAMD17-03-1-0469 TITLE: Effect of Chimaerins, Novel Receptors for Phorbol Esters, on Breast Cancer Cell Proliferation and Cell Cycle Progression PRINCIPAL INVESTIGATOR:

for AACR annual meeting 2006 Heregulin β1 induces cyclin D1 and p21 expression and promotes breastcancer cell proliferation through ErbB receptor/Rac/Erk Chengfeng Yang, Ying Liu, Mark A. Lemmon, and Marcelo G. Kazanietz. Department of Pharmacology, University of Pennsylvania School of Medicine,Philadelphia, PA 19104-6160 The human ErbB receptor family comprises 4 tyrosine kinase receptors (ErbB1or EGFR, ErbB2, ErbB3, and ErbB4). It is well established that dysregulation ofErbB receptor signaling plays important roles in the progression of various typesof cancers, including breast, prostate and colon cancer. Heregulins (also calledneuregulins) are a group of EGF-like ligands for the ErbB3 and ErbB4 receptorsand are often expressed in breast cancer tissues. In our previous studies, wefound that heregulin β1 (HRG) is a potent activator of Rac in breast cancer cells.The Rac activation by HRG is impaired by RNAi depletion of ErbB2 and ErbB3but not by ErbB4, and it also involves the transactivation of EGFR. In this study,we further determined how Rac activation by HRG mediates breast cancer T-47Dcell proliferation. We found that expression of β2-chimaerin, a Rac-GAP, or adominant negative Rac (N17Rac1) dose-dependently inhibited HRG-induced Racactivation in T-47D cells. Rac inhibition with β2-chimaerin and N17Rac1, or Rac1depletion using RNAi, dose-dependently inhibited HRG-induced cyclin D1 mRNAand protein expression and cell proliferation. Rac inhibition also impaired HRG-induced activation of Erk1/2. Inhibition of MEK with U0126 significantly reducedcyclin D1 induction and proliferation by HRG. HRG also induced rapid mRNA andprotein expression of p21 while it promotes cell proliferation. Rac inactivation ordepletion, or MEK inhibition impaired HRG-induced p21 up-regulation. Moreover,RNAi depletion of p21 impaired HRG-induced cell proliferation. RNAi depletion ofErbB3, ErbB2, or EGFR, but not ErbB4, also impaired HRG-induced cellproliferation. Together, these results indicate that heregulin β1 promotes breastcancer cell proliferation through ErbB receptor/Rac/Erk-dependent cyclin D1 andsuggests a paradoxical role for p21 in proliferation. (The U.S. Army MedicalResearch and Material Command under DAMD 17-03-1-0469 supported thiswork.) Abstract for AACR annual meeting 2005for AACR annual meeting 2005 Essential role for Rac1 in Heregulin β1-induced Mitogenic Signaling in Human BreastCancer Cells Chengfeng Yang, Ying Liu, and Marcelo G. Kazanietz Center for Experimental Therapeutics and Department of Pharmacology, University ofPennsylvania School of Medicine, Philadelphia, PA 19104-6160. The ErbB family of tyrosine kinase receptors comprises 4 members (EGFR orErbB1, Her2 or ErbB2, ErbB3 and ErbB4) which play important roles in the progressionof various types of cancers, including breast, prostate and colon cancer. Heregulin β1(HRG) belongs to the family of neuregulins, a group of peptide ligands for the ErbB3 andErbB4 receptors. How HRG causes the activation of PI3K-Akt and MAPKs to controlcell survival and proliferation is not fully understood. We explored whether Rho GTPasesplay critical roles in HRG-triggered mitogenic signaling. Using a PBD pull downapproach, we determined that HRG activates Rac1 in a doseand time-dependent mannerin MCF-7 and T-47D breast cancer cells. HRG-induced Rac1 activation showed astriking different kinetics from EGF-triggered activation of Rac1 in these two cell lines.While EGF-induced Rac1 activation peaked at 1-2 min and returned to basal within 15-30 min, HRG-triggered activation of Rac1 peaked at 5-10 min and still remained high 60min after stimulation. HRG also caused sustained activation of Cdc42 and RhoA with asimilar time-course. By using pharmacological inhibitors, specific blocking antibodiesand small interference RNA (siRNA) for individual ErbB receptors, it was determinedthat the activation of Rac1 by HRG is mediated by ErbB2 and ErbB3 receptors, and thatthere was an essential requirement for the EGFR in the HRG effect. HRG-induced Rac1activation was dependent on PI3K but not on Src, as it was impaired by wortmannin butnot by PP2. The kinetics of HRGand EGF-induced Rac1 activation strongly correlatedwith that for the activation of Erk1/2, JNK and p38 MAPKs. Inactivation of Rac1 byadenoviral delivery of beta2-chimaerin, a Rac-GAP, impaired HRG-induced activation ofMAPKs. Moreover, beta2-chimaerin, which did not affect Cdc42 and RhoA activation,also inhibited HRG-induced breast cancer cell proliferation. On the other hand,expression of a constitutively active Rac mutant (V12Rac1) in MCF-7 cells rescued theinhibitory effect of beta2-chimaerin on cell proliferation. These results suggest that Rac1is an important mediator of HRG mitogenic signaling in breast cancer cells and highlightthe complexity in HRG responses via activation of multiple tyrosine-kinase receptors(The U.S. Army Medical Research and Material Command under DAMD 17-03-1-0469supported this work.). Abstract for Era of Hope 05 DOD Breast Cancer Research Program Meetingfor Era of Hope 05 DOD Breast Cancer Research Program Meeting HEREGULIN β1-INDUCED RAC ACTIVATION PROMOTES BREASTCANCER CELL PROLIFERATION Chengfeng Yang, Ying Liu, and Marcelo G. KazanietzCenter for Experimental Therapeutics and Department of Pharmacology, University ofPennsylvania School of Medicine, Philadelphia, PA 19104-6160.E-mail: [email protected] Heregulin β1 (HRG) belongs to the family of neuregulins, a group of epidermal growthfactor (EGF)-like peptide ligands for the ErbB3 and ErbB4 receptors. It has been foundthat HRG1 is overexpressed in many kinds of human cancers including breast cancer, andthat it promotes breast cancer cell proliferation. The ErbB family of tyrosine kinasereceptors comprises 4 members (EGFR or ErbB1, Her2 or ErbB2, ErbB3 and ErbB4)which play important roles in the progression of various types of cancers including breastcancer. How HRG causes the activation of PI3K-Akt and mitogen-activated proteinkinases (MAPKs) to control breast cancer cell survival and proliferation is not fullyunderstood. We explored whether Rho GTPases play critical roles in HRG-triggeredmitogenic signaling. Recombinant HRG and two human breast cancer cell lines MCF-7 and T-47D were usedfor this study. Using a PBD pull down approach, we found that HRG activates Rac1 in adoseand time-dependent manner in MCF-7 and T-47D breast cancer cells. HRG-induced Rac1 activation showed a striking different kinetics from EGF-triggeredactivation of Rac1 in these two cell lines. While EGF-induced Rac1 activation peaked at1-2 min and returned to basal within 15-30 min, HRG-triggered activation of Rac1peaked at 5-10 min and still remained high 60 min after stimulation. HRG also causedsustained activation of Cdc42 and RhoA with a similar time-course. By usingpharmacological inhibitors, specific blocking antibodies and small interference RNA(siRNA) for individual ErbB receptors, it was determined that the activation of Rac1 byHRG is mediated by ErbB2 and ErbB3 receptors, and that there was an essentialrequirement for the EGFR in the HRG effect. HRG-induced Rac1 activation wasdependent on PI3K but not on Src, as it was impaired by wortmannin but not by PP2. Thekinetics of HRGand EGF-induced Rac1 activation strongly correlated with that for theactivation of Erk1/2, JNK and p38 MAPKs. Inactivation of Rac1 by adenoviral deliveryof beta2-chimaerin, a specific Rac-GAP, impaired HRG-induced activation of MAPKs.Moreover, beta2-chimaerin, which did not affect Cdc42 and RhoA activation, alsoinhibited HRG-induced breast cancer cell proliferation. On the other hand, expression ofa constitutively active Rac mutant (V12Rac1) in MCF-7 cells rescued the inhibitoryeffect of beta2-chimaerin on cell proliferation. We conclude that HRG-induced Rac activation is an important mediator of HRGmitogenic signaling in breast cancer cells. These results also indicate that approachesaimed at targeting Rac signaling could open new avenues for breast cancer therapeutics. The U.S. Army Medical Research and Material Command under DAMD 17-03-1-0469supported this work. Abstract for 20 annual meeting on oncogenes--2004for 20 annual meeting on oncogenes--2004 β2-Chimaerin Inhibits Heregulin-induced Activation of Mitogen-Activated Protein Kinases in Human Breast Cancer Cells Chengfeng Yang, Ying Liu, and Marcelo G. Kazanietz Center for Experimental Therapeutics and Department of Pharmacology,University of Pennsylvania School of Pennsylvania, Philadelphia, PA 19104 β2-Chimaerin is a “non-protein kinase C (PKC)” phorbol ester receptor. Previousstudies have shown that phorbol ester stimulation induces intracellular β2-chimaerin redistribution and interaction with other proteins. Its biological function,however, remains largely unknown. Heregulin is a specific ligand for ErbB3(Her3) and ErbB4 (Her4) receptors that are overexpressed in human breastcancers. Heregulin signaling is considered to play an important role in breastcarcinogenesis. Our preliminary study showed that the expression of β2-chimaerin in human breast cancer cells is greatly lower than that in humannormal breast cells. The present study is designed to investigate the effect of β2-chimaerin on heregulin-induced signal transduction and cell proliferation inhuman breast cancer MCF-7 and T-47 D cells. The overexpression of β2-chimaerin in breast cancer cells was achieved by adenoviral gene delivery. Theresults showed that treatment of breast cancer cells with heregulin-β1 resulted inthe activation of AKT, extracellular signal-regulated kinase (Erk), c-Jun NH(2)-terminal kinase (JNK) and cell proliferation in the absence of serum. Heregulin-induced AKT, Erk and JNK activation and cell growth were doseand time-dependent. Inhibition of phosphatidylinositol 3'-kinase (PI-3-K) activity withwortmannin completely blocked heregulin-induced AKT, Erk and JNK activationand cell growth. Overexpression of β2-chimaerin by adenoviral infection dose-dependently inhibited heregulin-triggered Erk and JNK, but not AKT activation.Moreover, overexpression of β2-chimaerin also does-dependently suppressedheregulin-induced cell proliferation as determined by BrdU incorporation. Theseresults suggest that β2-chimaerin impairs heregulin-initiated mitogenic signalingdownstream of PI-3-K through inhibition of Erk and JNK activity. (The U.S. ArmyMedical Research and Material Command under DAMD 17-03-1-0469 supportedthis work.)