A novel dual kinase function of the RET proto-oncogene negatively regulates activating transcription factor 4-mediated apoptosis.

The RET proto-oncogene, a tyrosine kinase receptor, is widely known for its essential role in cell survival. Germ line missense mutations, which give rise to constitutively active oncogenic RET, were found to cause multiple endocrine neoplasia type 2, a dominant inherited cancer syndrome that affects neuroendocrine organs. However, the mechanisms by which RET promotes cell survival and prevents cell death remain elusive. We demonstrate that in addition to cytoplasmic localization, RET is localized in the nucleus and functions as a tyrosine-threonine dual specificity kinase. Knockdown of RET by shRNA in medullary thyroid cancer-derived cells stimulated expression of activating transcription factor 4 (ATF4), a master transcription factor for stress-induced apoptosis, through activation of its target proapoptotic genes NOXA and PUMA. RET knockdown also increased sensitivity to cisplatin-induced apoptosis. We observed that RET physically interacted with and phosphorylated ATF4 at tyrosine and threonine residues. Indeed, RET kinase activity was required to inhibit the ATF4-dependent activation of the NOXA gene because the site-specific substitution mutations that block threonine phosphorylation increased ATF4 stability and activated its targets NOXA and PUMA. Moreover, chromatin immunoprecipitation assays revealed that ATF4 occupancy increased at the NOXA promoter in TT cells treated with tyrosine kinase inhibitors or the ATF4 inducer eeyarestatin as well as in RET-depleted TT cells. Together these findings reveal RET as a novel dual kinase with nuclear localization and provide mechanisms by which RET represses the proapoptotic genes through direct interaction with and phosphorylation-dependent inactivation of ATF4 during the pathogenesis of medullary thyroid cancer.

Staurosporine is chemoprotective by inducing G1 arrest in a Chk1- and pRb-dependent manner.

Chemotherapeutic agents have been the mainstay of cancer therapy for years. However, their effectiveness has been limited by toxicities they impart on normal cells. Staurosporine (ST) has been shown to arrest normal, but not breast cancer, cells in G1. Therefore, ST may become a chemoprotective agent, arresting normal cells while allowing tumor cells to enter cell cycle phases where they are sensitive to chemotherapeutic agents. Understanding the mechanism of ST-mediated G1 arrest may allow for a beneficial chemoprotective treatment strategy for patients. We utilized 76NE6 (pRb+/p53-), 76NF2V (pRb+/p53+) and 76NE7 (pRb-/P53+) non-tumorigenic human mammary epithelial cell lines to understand the role of the Rb and p53 pathways in ST-directed G1 arrest. CDK4 was downregulated by ST in Rb+ cells, but its presence could not reverse the arrest, neither did its stable downregulation alter ST-mediated cellular response. ST-mediated G1 arrest required pRb, which in turn initiated a cascade of events leading to inhibition of CDK4. Further assessment of this pathway revealed that Chk1 expression and activity were required for the Rb-dependent arrest. For example, pRb+ cells with small interfering RNA to Chk1 had approximately 60% less cells in G1 phase compared with controls and pRb- cells do not arrest upon ST. Furthermore, Chk1 expression facilitates the release of the Rb+ cells from G1 arrest. Collectively, our data suggest that pRb cooperates with Chk1 to mediate a G1 arrest only in pRb+ cells. The elucidation of this pathway can help identify novel agents to protect cancer patients against the debilitating effects of chemotherapy.

Dynein light chain 1, a p21-activated kinase 1-interacting substrate, promotes cancerous phenotypes.

We identified dynein light chain 1 (DLC1) as a physiologic substrate of p21-activated kinase 1 (Pak1). Pak1-DLC1 interaction plays an essential role in cell survival, which depends on Pak1's phosphorylation of DLC1 on Ser88. Pak1 associates with the complex of DLC1 and BimL, a proapoptotic BH3-only protein, and phosphorylates both proteins. Phosphorylation of BimL by Pak1 prevents it from interacting with and inactivation of Bcl-2, an antiapoptotic protein. Overexpression of DLC1 but not DLC1-Ser88Ala mutant promotes cancerous properties of breast cancer cells. DLC1 protein level is elevated in more than 90% of human breast tumors. The regulation of cell survival functions by Pak1-DLC1 interaction represents a novel mechanism by which a signaling kinase might regulate the cancerous phenotypes.

Osteocyte CIITA aggravates osteolytic bone lesions in myeloma.

Osteolytic destruction is a hallmark of multiple myeloma, resulting from activation of osteoclast-mediated bone resorption and reduction of osteoblast-mediated bone formation. However, the molecular mechanisms underlying the differentiation and activity of osteoclasts and osteoblasts within a myelomatous microenvironment remain unclear. Here, we demonstrate that the osteocyte-expressed major histocompatibility complex class II transactivator (CIITA) contributes to myeloma-induced bone lesions. CIITA upregulates the secretion of osteolytic cytokines from osteocytes through acetylation at histone 3 lysine 14 in the promoter of TNFSF11 (encoding RANKL) and SOST (encoding sclerostin), leading to enhanced osteoclastogenesis and decreased osteoblastogenesis. In turn, myeloma cell-secreted 2-deoxy-D-ribose, the product of thymidine catalyzed by the function of thymidine phosphorylase, upregulates CIITA expression in osteocytes through the STAT1/IRF1 signaling pathway. Our work thus broadens the understanding of myeloma-induced osteolysis and indicates a potential strategy for disrupting tumor-osteocyte interaction to prevent or treat patients with myeloma bone disease.

ATF4 loss of heterozygosity is associated with poor overall survival in medullary thyroid carcinoma.

Activating transcription factor 4 (ATF4) is a crucial mediator of the integrated stress response and a negative regulator of RET tyrosine kinase receptor in medullary thyroid carcinoma (MTC). However, the impact of genomic abnormalities in the ATF4 locus on MTC pathogenesis and response to tyrosine kinase inhibitor therapy remains unknown. Here, we evaluated ATF4 copy number variation and protein levels, with overall survival and response to TKIs in a clinical cohort of fifty-nine sporadic primary MTC. We assessed the somatic RETM918T mutation by sequencing, ATF4 copy number by a real-time polymerase chain reaction, and ATF4 protein levels using immunohistochemistry. This MTC cohort comprised 45 (76%) stage IV patients with a median follow-up of 100 months (interquartile range: 58-134 months). Somatic RETM918T was present in 23/57 (40%) tumors. Mono-allelic (36%; 21/59) and bi-allelic (5%; 3/59) loss of ATF4 was identified and was associated with low ATF4 protein expression (0-20%). Kaplan-Meier curves highlight low ATF4 protein or ATF4 loss alone had a significant negative impact on median survival compared to high protein expression (P<0.001) or diploid ATF4 (P=0.011), respectively. The combination of somatic RETM918T and low ATF4 protein levels further decreased overall survival. Both allelic loss and protein reduction were associated with worse overall survival (HR=3.79, 4.06 +RETM918T , and HR=10.64, 11.66 +RETM918T , respectively). Additionally, all 4 of the 11 patients treated with TKIs with a progressive disease by RECIST had low tumor ATF4 protein, with the two partial responder's tumors having high ATF4 protein. These findings suggest that ATF4 may predict response to tyrosine kinase inhibitors, serve as a prognostic marker for personalized care, and a therapeutic target in MTC.

ONC201 Shows Potent Anticancer Activity Against Medullary Thyroid Cancer via Transcriptional Inhibition of RET, VEGFR2, and IGFBP2.

Gain-of-function point mutations in the receptor tyrosine kinase RET, a driver oncogene in medullary thyroid carcinoma (MTC), prevent apoptosis through inhibition of ATF4, a critical transcriptional regulator of endoplasmic reticulum stress. However, the critical regulatory mechanisms driving RET-dependent oncogenesis remain elusive, and there is a clinical need to identify a transcriptional RET inhibitor. Here, we found that RET depletion decreased IGFBP2 and VEGFR2 mRNA and protein expression in MTC cells. IGFBP2 knockdown decreased cell survival and migration of MTC cells. In patients, IGFBP2 expression increased in metastatic MTC, and high IGFBP2 associated with poor overall survival. VEGFR2 protein levels were positively associated with RET expression in primary tumors, and VEGF-mediated increased cell viability was RET dependent. The small-molecule ONC201 treatment of MTC cells caused apoptotic cell death, decreased transcription of RET, VEGFR2, IGFBP2, increased mRNA levels of ATF4, and ATF4 target genes including DDIT3, BBC3, DUSP8, MKNK2, KLF9, LZTFL1, and SESN2 Moreover, IGFBP2 depletion increased ONC201-induced cell death. ONC201 inhibited tumor growth at a well-tolerated dose of 120 mg/kg/week administered by oral gavage and decreased MTC xenograft cell proliferation and angiogenesis. The protein levels of RET, IGFBP2, and VEGFR2 were decreased in ONC201-treated xenografts. Our study uncovered a novel ONC201 mechanism of action through regulation of RET and its targets, VEGFR2 and IGFBP2; this mechanism could be translated into the clinic and represent a promising strategy for the treatment of all patients with MTC, including those with TKI-refractory disease and other cancer with RET abnormalities.

RAC1 Alterations Induce Acquired Dabrafenib Resistance in Association with Anaplastic Transformation in a Papillary Thyroid Cancer Patient.

BRAF-activating mutations are the most frequent driver mutations in papillary thyroid cancer (PTC). Targeted inhibitors such as dabrafenib have been used in advanced BRAF-mutated PTC; however, acquired resistance to the drug is common and little is known about other effectors that may play integral roles in this resistance. In addition, the induction of PTC dedifferentiation into highly aggressive KRAS-driven anaplastic thyroid cancer (ATC) has been reported. We detected a novel RAC1 (P34R) mutation acquired during dabrafenib treatment in a progressive metastatic lesion with ATC phenotype. To identify a potential functional link between this novel mutation and tumor dedifferentiation, we developed a cell line derived from the metastatic lesion and compared its behavior to isogenic cell lines and primary tumor samples. Our data demonstrated that RAC1 mutations induce changes in cell morphology, reorganization of F-actin almost exclusively at the cell cortex, and changes in cell adhesion properties. We also established that RAC1 amplification, with or without mutation, is sufficient to drive cell proliferation and resistance to BRAF inhibition. Further, we identified polyploidy of chromosome 7, which harbors RAC1, in both the metastatic lesion and its derived cell line. Copy number amplification and overexpression of other genes located on this chromosome, such as TWIST1, EGFR, and MET were also detected, which might also lead to dabrafenib resistance. Our study suggests that polyploidy leading to increased expression of specific genes, particularly those located on chromosome 7, should be considered when analyzing aggressive thyroid tumor samples and in further treatments.

HEREDITARY ENDOCRINE TUMOURS: CURRENT STATE-OF-THE-ART AND RESEARCH OPPORTUNITIES: The state of science in medullary thyroid carcinoma: current challenges and unmet needs.

The 16th International Multiple Endocrine Neoplasia Workshop (MEN2019) held in Houston, TX, USA, focused on emerging topics in the pathogenesis and therapy of malignant endocrine tumors associated with MEN syndromes. With MEN-2 syndromes, the most common malignancy is medullary thyroid carcinoma (MTC). In the spirit of the original MEN meeting workshop model, the conference included didactic lectures and interactive working groups of clinicians and researchers focused on the state of science in MTC and ongoing challenges or unmet needs in the understanding of MTC and to develop strategies to address these issues.

Essential functions of p21-activated kinase 1 in morphogenesis and differentiation of mammary glands.

Although growth factors have been shown to influence mammary gland development, the nature of downstream effectors remains elusive. In this study, we show that the expression of p21-activated kinase (Pak)1, a serine/threonine protein kinase, is activated in mammary glands during pregnancy and lactation. By targeting an ectopic expression of a kinase-dead Pak1 mutant under the control of ovine beta-lactoglobulin promoter, we found that the mammary glands of female mice expressing kinase-dead Pak1 transgene revealed incomplete lobuloalveolar development and impaired functional differentiation. The expression of whey acidic protein and beta-casein and the amount of activated Stat5 in the nuclei of epithelial cells in transgenic mice were drastically reduced. Further analysis of the underlying mechanisms revealed that Pak1 stimulated beta-casein promoter activity in normal mouse mammary epithelial cells and also cooperated with Stat5a. Pak1 directly interacted with and phosphorylated Stat5a at Ser 779, and both COOH-terminal deletion containing Ser 779 of Stat5a and the Ser 779 to Ala mutation completely prevented the ability of Pak1 to stimulate beta-casein promoter. Mammary glands expressing inactive Pak1 exhibited a reduction of Stat5a Ser 779 phosphorylation. These findings suggest that Pak1 is required for alveolar morphogenesis and lactation function, and thus, identify novel functions of Pak1 in the mammary gland development.

Identification of Pax5 as a target of MTA1 in B-cell lymphomas.

Previously, we have shown that metastasis-associated protein 1 (MTA1) overexpression in transgenic mice was accompanied by high incidence of spontaneous B-cell lymphomas including diffuse large B-cell lymphomas (DLBCL). To understand the molecular basis of lymphoma in MTA1-transgenic (MTA1-TG) mice, we wished to identify a putative MTA1 target with a causal role in B-cell lymphogenesis. Using chromatin immunoprecipitation assays, we identified paired box gene 5 (Pax5), a molecule previously implicated in B-cell lymphogenesis, as a potential downstream effector of MTA1. Lymphomas from MTA1-TG mice also showed up-regulation of Pax5. We also found that MTA1 acetylated on Lys(626) interacted with p300 histone acetyltransferase, and that acetylated MTA1 was recruited to the Pax5 promoter to stimulate Pax5 transcription. Global gene profiling identified down-regulation of a set of genes, including those downstream of Pax5 and directly implicated in the B-cell lymphogenesis. Significance of these murine studies was established by evidence showing a widespread up-regulation of both MTA1 and Pax5 in DLBCL from humans. These observations provide in vivo genetic evidence for a role of MTA1 in lymphomagenesis.

Metastasis-associated protein 1 transgenic mice: a new model of spontaneous B-cell lymphomas.

Metastasis-associated protein 1 (MTA1), a component of the nuclear remodeling complex and the founding homologue of the MTA family, has been implicated in metastasis, but definitive causative evidence in an animal model system is currently lacking. Here, we show that MTA1 overexpression in transgenic mice is accompanied by a high incidence of spontaneous B cell lymphomas including diffuse large B cell lymphomas (DLBCL). Lymphocytes and lymphoma cells from MTA1-TG mice are hyperproliferative. Lymphomas were transplantable and of clonal origin and were characterized by down-regulation of p27Kip1 as well as up-regulation of Bcl2 and cyclin D1. The significance of these murine studies was established by evidence showing a widespread up-regulation of MTA1 in DLBCL from humans. These findings reveal a previously unrecognized role for the MTA1 pathway in the development of spontaneous B cell lymphomas, and offer a potential therapeutic target in B cell lymphomas. These observations suggest that MTA1-TG mice represent a new model of spontaneous DLBCL associated with high tumor incidence and could be used for therapeutic intervention studies.

Combinations of Tyrosine Kinase Inhibitor and ERAD Inhibitor Promote Oxidative Stress-Induced Apoptosis through ATF4 and KLF9 in Medullary Thyroid Cancer.

Medullary thyroid carcinoma (MTC) originates from the C cells of the thyroid gland, which secrete calcitonin. Lymph node and distant metastases are frequently present at diagnosis. Activating mutations of RET, a driver oncogene in MTC that encodes a tyrosine kinase receptor, prevents apoptosis through inhibition of ATF4, a key transcriptional regulator of endoplasmic reticulum (ER) stress. We hypothesized that the combination of a tyrosine kinase inhibitor (TKI) and an ATF4 inducer promotes cell death by triggering catastrophic oxidative stress and apoptotic cell death. Here, we report that the ER-associated protein degradation (ERAD) inhibitor eeyarestatin sensitized MTC cells to the TKIs, sunitinib and vandetanib, thereby leading to synergistic upregulation of ATF4 expression, accumulation of reactive oxygen species, and subsequent cell death. Genome-wide analysis of ATF4 interaction sites by chromatin immunoprecipitation (ChIP) sequencing revealed that among ATF4 target genes was KLF9 (Kruppel-like factor 9), which induces MTC apoptosis. ChIP assays revealed that ATF4 occupancy at the KLF9 promoter was increased in MTC cells treated with eeyarestatin or vandetanib alone and was further enhanced in cells treated with both drugs, leading to increased KLF9 transcription. Depletion of ATF4 by shRNA led to downregulation of KLF9 expression and prevented oxidative stress-induced cell death. Furthermore, we identified ATF4 target genes (LZTFL1, MKNK2, and SIAH1 with known tumor suppressor function) that were synergistically upregulated with the combination of TKI and ERAD inhibitor. IMPLICATIONS: These findings reveal a combination therapy that induces reactive oxygen species-dependent catastrophic cell death through induction of ATF4 and KLF9 transcriptional activity.

Oncogenic and osteolytic functions of histone demethylase NO66 in castration-resistant prostate cancer.

Epigenetic changes that cause dysregulated gene expression during progression of androgen-independent prostate cancer (PCa) and metastatic skeletal lesions remain elusive. Here, we explored the role of histone demethylase NO66 in the pathogenesis of PCa and bone metastasis-related skeletal lesions. Tissue and cDNA microarrays of PCa were analyzed for NO66 mRNA and protein levels. We examined the effects of gain and loss of NO66 function on cell viability, colony formation, migration, invasion, and tumor-induced skeletal lesions in femoral bone. RNAseq and ChIPseq were performed to elucidate NO66-target genes in PCa. We report that NO66 levels were upregulated in advanced primary prostate tumors compared to normal tissue or tumors with low Gleason scores. Forced expression of NO66 promoted cell survival and invasion of PCa cells; whereas, knockdown of NO66 resulted in decreased cell survival and increased sensitivity to docetaxel. NO66-overexpressing PC3 cells implanted into the femoral bone of male SCID mice caused massive bone loss and stimulation of mouse osteoclast-promoting genes, including Dickkopf1, Cathepsin K, Nf-kß,; and Calcr, suggesting a role for NO66 in tumor growth in bone and osteoclast activity. Combined RNAseq and ChIP-seq revealed that NO66 activates the survival gene MCL1, the invasion-associated genes IGFBP5 and MMP3, the pro-oncogenic genes CTNNB1 and CCND1, and the epigenetic modifier gene KMT2A in androgen-independent PCa. Our findings uncover the role of NO66 as a key oncogenic driver in PCa, causing osteolytic lesions through upstream epigenetic regulation of key genes for survival, invasion and metastasis, and pro-osteoclastic factors.

ATF4 Targets RET for Degradation and Is a Candidate Tumor Suppressor Gene in Medullary Thyroid Cancer.

Context: Medullary thyroid cancer (MTC) is an aggressive tumor that harbors activating mutations of the RET proto-oncogene. We previously reported that RET inhibits transcriptional activity of ATF4, the master regulator of the stress response pathway, to prevent cell death. Objective: We hypothesized that loss of function of ATF4 plays a role in initiation of MTC. Design: Targeted deletion of Atf4 in mice was used to assess ATF4 function in the thyroid gland. ATF4 overexpression was achieved by adenoviral and lentiviral vectors. We used immunohistochemical analysis and western blotting of MTC tumors to determine protein levels of RET and ATF4 and the Kaplan-Meier method to determine their association with clinical outcome. Results: Targeted deletion of Atf4 in mice causes C-cell hyperplasia, a precancerous lesion for MTC. Forced ATF4 expression decreased survival of MTC cells and blocked the activation of RET downstream signaling pathways (phosphorylated ERK, phosphorylated AKT, and p70S6K). ATF4 knockdown decreased sensitivity to tyrosine kinase inhibitor-induced apoptosis. Moreover, ATF4 expression decreased RET protein levels by promoting RET ubiquitination. We found decreased or loss of ATF4 in 52% of MTC tumors (n = 39) compared with normal thyroid follicle cells. A negative correlation was observed between RET and ATF4 protein levels in MTC tumors, and low ATF4 expression was associated with poor overall survival in patients with MTC. Conclusions: ATF4 was identified as a negative regulator of RET, a candidate tumor suppressor gene, and may be a molecular marker that distinguishes patients at high risk of MTC from those with a longer survival prognosis.

Antihuman epidermal growth factor receptor 2 antibody herceptin inhibits autocrine motility factor (AMF) expression and potentiates antitumor effects of AMF inhibitors.

Overexpression of the human epidermal growth factor receptor (HER) 2 has been linked to the development and maintenance of malignant phenotypes in breast tumors. In addition, the growth and dissemination of human cancers are regulated in part by the autocrine motility factor (AMF)/phosphoglucose isomerase shown to be up-regulated by heregulin (HRG) in breast cancer cells. This study was undertaken to explore the effect of anti-HER2 monoclonal antibody 4D5 [Herceptin (HCT)] on AMF expression and the potential of its augmentation by specific simple sugar AMF inhibitors. Here we show that HCT treatment of high HER2-expressing breast cancer SK-BR3, BT-474, and ZR-75R cells resulted in down-regulation of AMF mRNA and protein. HCT inhibited the ability of HRG to induce AMF expression in cells with a normal HER2 level, and HCT-mediated down-regulation could be reversed by HRG treatment in breast cancer cells with a high HER2 level. HCT also inhibited transcription from a chimeric pGL3-Luc vector-based reporter system containing the 1.8-kb promoter region of human AMF. Treatment of breast cancer cells with the combination of HCT and specific AMF inhibitors, erythrose 4-phosphate or D-mannose 6-phosphate, resulted in an additive inhibitory effect on both the growth rate and invasiveness of cells as compared with treatment with each agent alone. Results presented here suggest that HCT can effectively block both ligand-induced and constitutive expression of AMF associated with high HER2 overexpression, implying a role of the AMF pathway in the action of HCT. Accordingly, the combination of AMF inhibitor with HCT can potentiate the growth-inhibitory and anti-invasive action of HCT in breast cancer cells.

HER2 regulation of peroxisome proliferator-activated receptor gamma (PPARgamma) expression and sensitivity of breast cancer cells to PPARgamma ligand therapy.

Induction of terminal differentiation of cancer cells is an evolving novel therapeutic approach, and accordingly, peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-stimulated transcription factor with differentiation-promoting activity and overexpressed in a variety of cancers, has emerged as one of the promising therapeutic targets. Because c-erbB family growth factor receptor 2 (HER2) overexpression is one of the most recognizable molecular dysfunctions in breast tumors, in the studies presented here, we explored the effect of HER2 overexpression on the status of PPARgamma expression and on the sensitivity of breast cancer cells to PPARgamma-ligand troglitazone-induced growth inhibition. We show that HER2 overexpression in MCF7 breast cancer cells enhanced the expression of PPARgamma-mRNA and -protein. Furthermore, PPARgamma expression was dramatically increased in 11 of 16 breast tumors as compared with the adjacent normal tissue. In addition, HER2 up-regulation resulted in a partial inhibition of transcriptional activity of the endogenous PPARgamma, stimulation to differentiation, and resistance to troglitazone-mediated inhibition of anchorage-independent growth of breast cancer cells. Conversely, down-regulation of HER2 by anti-HER2 monoclonal antibody Herceptin led to a decreased level of PPARgamma protein and sensitization of breast cancer cells to the inhibitory effects of troglitazone. In summary, these findings show for the first time that HER2 up-regulates PPARgamma expression and modulates the sensitivity of breast cancer cells to PPARgamma ligand therapy.

The epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 (Iressa) suppresses c-Src and Pak1 pathways and invasiveness of human cancer cells.

PURPOSE: Abnormalities in the expression and signaling pathways downstream of the epidermal growth factor receptor (EGFR) contribute to the progression, invasion, and maintenance of the malignant phenotype in human cancers, including those of the head and neck and breast. Accordingly, agents such as the EGFR tyrosine kinase inhibitor (EGFR-TKI) ZD1839 (Iressa) are promising, biologically based treatments that are in various stages of preclinical and clinical development. The process of tumor progression requires, among other steps, increased transformation, directional migration, and enhanced cell survival; this study explored the effect of ZD1839 on the stimulation of c-Src and p21-activated kinase 1 (Pak1), which are vital for transformation, directional motility, and cell survival of cancer cells. EXPERIMENTAL DESIGN: We examined the effect of ZD1839 on biochemical and functional assays indicative of directional motility and cell survival, using human head and neck squamous cancer cells and breast cancer cells. RESULTS: ZD1839 effectively inhibited c-Src activation and Pak1 activity in exponentially growing cancer cells. In addition, ZD1839 suppressed EGF-induced stimulation of EGFR autophosphorylation on Y1086 and Grb2-binding Y1068 sites, c-Src phosphorylation on Y215, and Pak1 activity. ZD1839 also blocked EGF-induced cytoskeleton remodeling, redistribution of activated EGFR, and in vitro invasiveness of cancer cells. CONCLUSIONS: These studies suggest that the EGFR-TKI ZD1839 may cause potent inhibition of the Pak1 and c-Src pathways and, therefore, have potential to affect the invasiveness of human cancer cells deregulated in these growth factor receptor pathways.

Suppression of epidermal growth factor receptor, mitogen-activated protein kinase, and Pak1 pathways and invasiveness of human cutaneous squamous cancer cells by the tyrosine kinase inhibitor ZD1839 (Iressa).

Abnormalities in the expression and signaling pathways downstream of the epidermal growth factor receptor (EGFR) contribute to malignant transformation in human cancers, including those of the cutaneous epithelium. Accordingly, novel agents such as the EGFR tyrosine kinase inhibitor ZD1839 (Iressa), are promising, biologically based treatments that are currently in preclinical and clinical development. The process of tumor progression requires, among other steps, increased transformation, directional migration, and enhanced cell survival. This study explored the effect of ZD1839 on the stimulation of p42/44 mitogen-activated protein kinase (MAPK) and p21-activated kinase 1 (Pak1), which are vital for transformation, directional motility, and cell survival, using immortalized keratinocytes (HaCaT cells) and cutaneous squamous cell carcinoma cells. The EGFR and a number of effector kinases (mitogen-activated protein extracellular signal-regulated kinase kinase 1 and 2, MAPK, Pak1, p38, c-JunNH(2)-terminal kinase and extracellular signal-regulated kinase 1) and cell survival proteins (AKT, FKHR, and c-Src) showed constitutive pathway activation in HaCaT and cutaneous squamous cell carcinoma cells. ZD1839 effectively inhibited EGFR and MAPK activation and Pak1 activity in exponentially growing cancer cells. ZD1839 also suppressed EGF-induced stimulation of EGFR autophosphorylation on Y1086 and Y1068, MAPK phosphorylation on T402 and Y404, and Pak1 activity in a dose-dependent manner. In addition, ZD1839 blocked EGF-induced cytoskeleton remodeling, cell growth, and in vitro invasiveness of cancer cells and induced a differentiated squamous cell phenotype. These studies suggest that the EGFR-tyrosine kinase inhibitor ZD1839 may cause potent inhibition of the EGFR, MAPK, and Pak1 pathways, resulting in attenuation of transformed cell phenotypes and induced differentiation in human cancer cells deregulated in these growth factor receptor pathways.

Emerging roles of MTA family members in human cancers.

Metastasis-associated genes (MTAs) represent a rapidly growing novel gene family. At present, there are three different known genes (MTA1, MTA2, and MTA3) and six reported isoforms (MTA1, MTA1s, MTA1-ZG29p, MTA2, MTA3, MTA3L). MTA1, MTA2, and MTA3 are components of the nucleosome remodeling and deacetylation complex, which is associated with adenosine triphosphate-dependent chromatin remodeling and transcriptional regulation. MTA proteins, as a part of the NuRD complex (nuclear remodeling and deacetylation complex), are thought to modulate transcription by influencing the status of chromatin remodeling. MTA1 overexpression is closely correlated with an aggressive course in several human carcinomas. Recent studies have shown that growth factor stimulation of breast cancer cells induces the expression of MTA1 and its interaction with and repression of the estrogen receptor (ER) transactivation function, leading to enhanced anchorage-independent growth in vitro and hormone independence. Furthermore, the status of the ER pathway modulates the expression of MTA3 as well as epithelial-to-mesenchymal transition in human breast tumors. MTA1 expression is not restricted to tumors; however, several normal mouse tissues and organs also express substantial levels of MTA1. Thus, MTA1 may play a role in both the physiologic and the pathologic states of cells. In Caenorhabditis elegans, MTA1-like genes regulate cell polarity, migration, embryonic patterning, and vulva development. In addition, two naturally occurring variants of MTA1, MTA1-ZG29p, and MTA1s have also been identified. ZG29p is an N-terminal truncated form of MTA1 and is present in the zymogen granules of the pancreas. In contrast, MTA1s is the C-terminal truncated form present in the cytoplasm. MTA1s binds and inhibits the nuclear functions of the ER by sequestering it to cytoplasm, stimulating the mitogen-activated protein kinase pathway. Furthermore, breast tumors with no or low ER in the nucleus exhibit elevated levels of MTA1s and cytoplasmic subcellular localization of the ER. This article reviews the current status of MTA biochemistry and its implications for tumor biology.

Cyclin E deregulation impairs mitotic progression through premature activation of Cdc25C.

The cyclin E-cyclin-dependent kinase 2 (CDK2) complex accelerates entry into the S phase of the cell cycle and promotes polyploidy, which may contribute to genomic instability in cancer cells. The effect of low molecular weight isoforms of cyclin E (LMW-E) overexpression on mitotic progression and its link to genomic instability were the focus of this study. Here, we show that full-length cyclin E (EL) and LMW-E overexpression impairs the G(2)-M transition differently by targeting dual-specificity phosphatase Cdc25C activity. We identify Cdc25C as an interaction partner and substrate for cyclin E/CDK2 kinase. Specifically, the cyclin E/CDK2 complex phosphorylates Cdc25C on Ser(214), leading to its premature activation, which coincides with higher cyclin B/CDK1 and Polo-like kinase 1 (PLK1) activities in an S-phase-enriched population that result in faster mitotic entry. Whereas EL overexpression leads to hyperactivation of Cdc25C, cyclin B/CDK1, and PLK1 in a G(2)-M-enriched population, LMW-E overexpression causes premature inactivation of Cdc25C and PLK1, leading to faster mitotic exit. In addition, LMW-E-overexpressing cells showed a reduction in the mitotic index in the presence of a spindle poison and faster degradation of cyclin B, suggesting an increased rate of mitotic slippage and adaptation to the spindle checkpoint. Lastly, downregulation of Cdc25C inhibits LMW-E-mediated chromosome missegregation, anaphase bridges, and centrosome amplification. These results suggest that the high levels of LMW-E isoforms found in breast cancer may contribute to cellular transformation and genomic instability by impairing mitotic progression involving Cdc25C.