Cynthia Lopez-Haber

Identification of the Rac-GEF P-Rex1 as an Essential Mediator of ErbB Signaling in Breast Cancer

While the small GTPase Rac1 and its effectors are well-established mediators of mitogenic and motile signaling by tyrosine kinase receptors and have been implicated in breast tumorigenesis, little is known regarding the exchange factors (Rac-GEFs) that mediate ErbB receptor responses. Here, we identify the PIP(3)-Gβγ-dependent Rac-GEF P-Rex1 as an essential mediator of Rac1 activation, motility, cell growth, and tumorigenesis driven by ErbB receptors in breast cancer cells. Notably, activation of P-Rex1 in breast cancer cells requires the convergence of inputs from ErbB receptors and a Gβγ- and PI3Kγ-dependent pathway. Moreover, we identified the GPCR CXCR4 as a crucial mediator of P-Rex1/Rac1 activation in response to ErbB ligands. P-Rex1 is highly overexpressed in human breast cancers and their derived cell lines, particularly those with high ErbB2 and ER expression. In addition to the prognostic and therapeutic implications, our findings reveal an ErbB effector pathway that is crucial for breast cancer progression.

Rac signaling in breast cancer: A tale of GEFs and GAPs

Rac GTPases, small G-proteins widely implicated in tumorigenesis and metastasis, transduce signals from tyrosine-kinase, G-protein-coupled receptors (GPCRs), and integrins, and control a number of essential cellular functions including motility, adhesion, and proliferation. Deregulation of Rac signaling in cancer is generally a consequence of enhanced upstream inputs from tyrosine-kinase receptors, PI3K or Guanine nucleotide Exchange Factors (GEFs), or reduced Rac inactivation by GTPase Activating Proteins (GAPs). In breast cancer cells Rac1 is a downstream effector of ErbB receptors and mediates migratory responses by ErbB1/EGFR ligands such as EGF or TGFα and ErbB3 ligands such as heregulins. Recent advances in the field led to the identification of the Rac-GEF P-Rex1 as an essential mediator of Rac1 responses in breast cancer cells. P-Rex1 is activated by the PI3K product PIP3 and Gβγ subunits, and integrates signals from ErbB receptors and GPCRs. Most notably, P-Rex1 is highly overexpressed in human luminal breast tumors, particularly those expressing ErbB2 and estrogen receptor (ER). The P-Rex1/Rac signaling pathway may represent an attractive target for breast cancer therapy.

Non-Small Cell Lung Carcinoma Cell Motility, Rac Activation and Metastatic Dissemination Are Mediated by Protein Kinase C Epsilon

Background Protein kinase C (PKC) ε, a key signaling transducer implicated in mitogenesis, survival, and cancer progression, is overexpressed in human primary non-small cell lung cancer (NSCLC). The role of PKCε in lung cancer metastasis has not yet been established. Principal Findings Here we show that RNAi-mediated knockdown of PKCε in H358, H1299, H322, and A549 NSCLC impairs activation of the small GTPase Rac1 in response to phorbol 12-myristate 13-acetate (PMA), serum, or epidermal growth factor (EGF). PKCε depletion markedly impaired the ability of NSCLC cells to form membrane ruffles and migrate. Similar results were observed by pharmacological inhibition of PKCε with εV1-2, a specific PKCε inhibitor. PKCε was also required for invasiveness of NSCLC cells and modulated the secretion of extracellular matrix proteases and protease inhibitors. Finally, we found that PKCε-depleted NSCLC cells fail to disseminate to lungs in a mouse model of metastasis. Conclusions Our results implicate PKCε as a key mediator of Rac signaling and motility of lung cancer cells, highlighting its potential as a therapeutic target.

Cucurbitacin I Inhibits Rac1 Activation in Breast Cancer Cells by a Reactive Oxygen Species-Mediated Mechanism and Independently of Janus Tyrosine Kinase 2 and P-Rex1

The small GTPase Rac1 has been widely implicated in mammary tumorigenesis and metastasis. Previous studies established that stimulation of ErbB receptors in breast cancer cells activates Rac1 and enhances motility via the Rac-guanine nucleotide exchange factor P-Rex1. As the Janus tyrosine kinase 2 (Jak2)/signal transducer and activator of transcription 3 (Stat3) pathway has been shown to be functionally associated with ErbB receptors, we asked if this pathway could mediate P-Rex1/Rac1 activation in response to ErbB ligands. Here we found that the anticancer agent cucurbitacin I, a Jak2 inhibitor, reduced the activation of Rac1 and motility in response to the ErbB3 ligand heregulin in breast cancer cells. However, Rac1 activation was not affected by Jak2 or Stat3 RNA interference, suggesting that the effect of cucurbitacin I occurs through a Jak2-independent mechanism. Cucurbitacin I also failed to affect the activation of P-Rex1 by heregulin. Subsequent analysis revealed that cucurbitacin I strongly activates RhoA and the Rho effector Rho kinase (ROCK) in breast cancer cells and induces the formation of stress fibers. Interestingly, disruption of the RhoA-ROCK pathway prevented the inhibitory effect of cucurbitacin I on Rac1 activation by heregulin. Lastly, we found that RhoA activation by cucurbitacin I is mediated by reactive oxygen species (ROS). The ROS scavenger N-acetyl l-cysteine and the mitochondrial antioxidant Mito-TEMPO rescued the inhibitory effect of cucurbitacin I on Rac1 activation. In conclusion, these results indicate that ErbB-driven Rac1 activation in breast cancer cells proceeds independently of the Jak2 pathway. Moreover, they established that the inhibitory effect of cucurbitacin I on Rac1 activity involves the alteration of the balance between Rho and Rac.

Differential regulation of gene expression by protein kinase C isozymes as determined by genome-wide expression analysis.

Protein kinase C (PKC) isozymes are key signal transducers involved in normal physiology and disease and have been widely implicated in cancer progression. Despite our extensive knowledge of the signaling pathways regulated by PKC isozymes and their effectors, there is essentially no information on how individual members of the PKC family regulate gene transcription. Here, we report the first PKC isozyme-specific analysis of global gene expression by microarray using RNAi depletion of diacylglycerol/phorbol ester-regulated PKCs. A thorough analysis of this microarray data revealed unique patterns of gene expression controlled by PKCα, PKCδ, and PKCϵ, which are remarkably different in cells growing in serum or in response to phorbol ester stimulation. PKCδ is the most relevant isoform in controlling the induction of genes by phorbol ester stimulation, whereas PKCϵ predominantly regulates gene expression in serum. We also established that two PKCδ-regulated genes, FOSL1 and BCL2A1, mediate the apoptotic effect of phorbol esters or the chemotherapeutic agent etoposide in prostate cancer cells. Our studies offer a unique opportunity for establishing novel transcriptional effectors for PKC isozymes and may have significant functional and therapeutic implications.

Transcriptional Regulation of Oncogenic Protein Kinase Cϵ (PKCϵ) by STAT1 and Sp1 Proteins

Background: PKCϵ, a kinase widely implicated in tumorigenesis and metastasis, is overexpressed in many cancers. Results: Transcription factors Sp1 and STAT1 control the expression of PKCϵ in cancer cells. Conclusion: Up-regulation of PKCϵ is mediated by dysregulated transcriptional mechanisms. Significance: Our results may have significant implications for the development of approaches to target PKCϵ and its effectors in cancer therapeutics. Overexpression of PKCϵ, a kinase associated with tumor aggressiveness and widely implicated in malignant transformation and metastasis, is a hallmark of multiple cancers, including mammary, prostate, and lung cancer. To characterize the mechanisms that control PKCϵ expression and its up-regulation in cancer, we cloned an ∼1.6-kb promoter segment of the human PKCϵ gene (PRKCE) that displays elevated transcriptional activity in cancer cells. A comprehensive deletional analysis established two regions rich in Sp1 and STAT1 sites located between −777 and −105 bp (region A) and −921 and −796 bp (region B), respectively, as responsible for the high transcriptional activity observed in cancer cells. A more detailed mutagenesis analysis followed by EMSA and ChIP identified Sp1 sites in positions −668/−659 and −269/−247 as well as STAT1 sites in positions −880/−869 and −793/−782 as the elements responsible for elevated promoter activity in breast cancer cells relative to normal mammary epithelial cells. RNAi silencing of Sp1 and STAT1 in breast cancer cells reduced PKCϵ mRNA and protein expression, as well as PRKCE promoter activity. Moreover, a strong correlation was found between PKCϵ and phospho-Ser-727 (active) STAT1 levels in breast cancer cells. Our results may have significant implications for the development of approaches to target PKCϵ and its effectors in cancer therapeutics.

PKCε IS AN ESSENTIAL MEDIATOR OF PROSTATE CANCER BONE METASTASIS

The bone is a preferred site for metastatic homing of prostate cancer cells. Once prostate cancer patients develop skeletal metastases, they eventually succumb to the disease; therefore, it is imperative to identify key molecular drivers of this process. This study examines the involvement of protein kinase C epsilon (PKCϵ), an oncogenic protein that is abnormally overexpressed in human tumor specimens and cell lines, on prostate cancer cell bone metastasis. PC3-ML cells, a highly invasive prostate cancer PC3 derivative with bone metastatic colonization properties, failed to induce skeletal metastatic foci upon inoculation into nude mice when PKCϵ expression was silenced using shRNA. Interestingly, while PKCϵ depletion had only marginal effects on the proliferative, adhesive, and migratory capacities of PC3-ML cells in vitro or in the growth of xenografts upon s.c. inoculation, it caused a significant reduction in cell invasiveness. Notably, PKCϵ was required for transendothelial cell migration (TEM) as well as for the growth of PC3-ML cells in a bone biomimetic environment. At a mechanistic level, PKCϵ depletion abrogates the expression of IL1β, a cytokine implicated in skeletal metastasis. Taken together, PKCϵ is a key factor for driving the formation of bone metastasis by prostate cancer cells and is a potential therapeutic target for advanced stages of the disease. Implications: This study uncovers an important new function of PKCϵ in the dissemination of cancer cells to the bone; thus, highlighting the promising potential of this oncogenic kinase as a therapeutic target for skeletal metastasis. Mol Cancer Res; 13(9); 1336–46. ©2015 AACR.

Heregulin/ErbB3 Signaling Enhances CXCR4-Driven Rac1 Activation and Breast Cancer Cell Motility via Hypoxia-Inducible Factor 1α

ABSTRACT The growth factor heregulin (HRG), a ligand of ErbB3 and ErbB4 receptors, contributes to breast cancer development and the promotion of metastatic disease, and its expression in breast tumors has been associated with poor clinical outcome and resistance to therapy. In this study, we found that breast cancer cells exposed to sustained HRG treatment show markedly enhanced Rac1 activation and migratory activity in response to the CXCR4 ligand SDF-1/CXCL12, effects mediated by P-Rex1, a Rac-guanine nucleotide exchange factor (GEF) aberrantly expressed in breast cancer. Notably, HRG treatment upregulates surface expression levels of CXCR4, a G protein-coupled receptor (GPCR) implicated in breast cancer metastasis and an indicator of poor prognosis in breast cancer patients. A detailed mechanistic analysis revealed that CXCR4 upregulation and sensitization of the Rac response/motility by HRG are mediated by the transcription factor hypoxia-inducible factor 1α (HIF-1α) via ErbB3 and independently of ErbB4. HRG caused prominent induction in the nuclear expression of HIF-1α, which transcriptionally activates the CXCR4 gene via binding to a responsive element located in positions −1376 to −1372 in the CXCR4 promoter, as revealed by mutagenesis analysis and chromatin immunoprecipitation (ChIP). Our results uncovered a novel function for ErbB3 in enhancing breast cancer cell motility and sensitization of the P-Rex1/Rac1 pathway through HIF-1α-mediated transcriptional induction of CXCR4.

The P-Rex1/Rac signaling pathway as a point of convergence for HER/ErbB receptor and GPCR responses

ABSTRACT Guanine nucleotide Exchange Factors (GEFs) are responsible for mediating GDP/GTP exchange for specific small G proteins, such as Rac. There has been substantial evidence for the involvement of Rac-GEFs in the control of cancer cell migration and metastatic progression. We have previously established that the Rac-GEF P-Rex1 is a mediator of actin cytoskeleton rearrangements and cell motility in breast cancer cells downstream of HER/ErbB receptors and the G-Protein Coupled Receptor (GPCR) CXCR4. P-Rex1 is highly expressed in luminal A and B breast cancer compared to normal mammary tissue, whereas expression is very low in basal breast cancer, and its expression correlates with the appearance of metastasis in patients. Here, we discuss the involvement of P-Rex1 as an effector of oncogenic/metastatic receptors in breast cancer and underscore its relevance in the convergence of receptor-triggered motile signals. In addition, we provide an overview of our recent findings describing a cross-talk between HER/ErbB receptors and CXCR4, and how this impacts on the activation of P-Rex1/Rac1 signaling, as well as highlight challenges that lie ahead. We propose a model in which P-Rex1 acts as a crucial node for the integration of upstream inputs from HER/ErbB receptors and CXCR4 in luminal breast cancer cells.

Discovery of a small-molecule protein kinase Cδ-selective activator with promising application in colon cancer therapy

Protein kinase C (PKC) isozymes play major roles in human diseases, including cancer. Yet, the poor understanding of isozymes-specific functions and the limited availability of selective pharmacological modulators of PKC isozymes have limited the clinical translation of PKC-targeting agents. Here, we report the first small-molecule PKCδ-selective activator, the 7α-acetoxy-6β-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz), which binds to the PKCδ-C1-domain. Roy-Bz potently inhibited the proliferation of colon cancer cells by inducing a PKCδ-dependent mitochondrial apoptotic pathway involving caspase-3 activation. In HCT116 colon cancer cells, Roy-Bz specifically triggered the translocation of PKCδ but not other phorbol ester responsive PKCs. Roy-Bz caused a marked inhibition in migration of HCT116 cells in a PKCδ-dependent manner. Additionally, the impairment of colonosphere growth and formation, associated with depletion of stemness markers, indicate that Roy-Bz also targets drug-resistant cancer stem cells, preventing tumor dissemination and recurrence. Notably, in xenograft mouse models, Roy-Bz showed a PKCδ-dependent antitumor effect, through anti-proliferative, pro-apoptotic, and anti-angiogenic activities. Besides, Roy-Bz was non-genotoxic, and in vivo it had no apparent toxic side effects. Collectively, our findings reveal a novel promising anticancer drug candidate. Most importantly, Roy-Bz opens the way to a new era on PKC biology and pharmacology, contributing to the potential redefinition of the structural requirements of isozyme-selective agents, and to the re-establishment of PKC isozymes as feasible therapeutic targets in human diseases.