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Synergistic cytotoxic effects of combination of eeyarestatin with sunitinib in MZCRC1 cells.

Functional categ ories of ATF4 target genes.

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.

Supplementary Movie 3 from Low Molecular Weight Cyclin E Overexpression Shortens Mitosis, Leading to Chromosome Missegregation and Centrosome Amplification

Supplementary Movie 5 from Low Molecular Weight Cyclin E Overexpression Shortens Mitosis, Leading to Chromosome Missegregation and Centrosome Amplification

Supplementary Movie 2 from Low Molecular Weight Cyclin E Overexpression Shortens Mitosis, Leading to Chromosome Missegregation and Centrosome Amplification

Supplementary Figure Legends 1-2, Table 1 Legend, Movie Legends 1-6 from Low Molecular Weight Cyclin E Overexpression Shortens Mitosis, Leading to Chromosome Missegregation and Centrosome Amplification

Supplementary Tables 1-4, Figures 1-4, Legends and Methods from Metastasis-Associated Protein 1 Transgenic Mice: A New Model of Spontaneous B-Cell Lymphomas

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 G2-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 Ser214, 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 G2-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. Cancer Res; 70(12); 5085–95. ©2010 AACR.

Supplementary Movie 1 from Low Molecular Weight Cyclin E Overexpression Shortens Mitosis, Leading to Chromosome Missegregation and Centrosome Amplification

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 Lys626 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. [Cancer Res 2007;67(15):7132–8]

Supplementary Figures 1-2, Tables 1-9 from Identification of Pax5 as a Target of MTA1 in B-Cell Lymphomas

Supplementary Figure 1 from Low Molecular Weight Cyclin E Overexpression Shortens Mitosis, Leading to Chromosome Missegregation and Centrosome Amplification

Supplementary Figure 1 Legend from Cyclin E Deregulation Impairs Mitotic Progression through Premature Activation of Cdc25C

Supplementary Figure 2 from Low Molecular Weight Cyclin E Overexpression Shortens Mitosis, Leading to Chromosome Missegregation and Centrosome Amplification

Overexpression of the low molecular weight isoforms (LMW-E) of cyclin E induces chromosome instability; however, the degree to which these tumor-specific forms cause genomic instability differs from that of full-length cyclin E (EL), and the underlying mechanism(s) has yet to be elucidated. Here, we show that EL and LMW-E overexpression impairs the G2-M transition differently and leads to different degrees of chromosome instability in a breast cancer model system. First, the most significant difference is that EL overexpression prolongs cell cycle arrest in prometaphase, whereas LMW-E overexpression reduces the length of mitosis and accelerates mitotic exit. Second, LMW-E–overexpressing cells are binucleated or multinucleated with amplified centrosomes, whereas EL-overexpressing cells have the normal complement of centrosomes. Third, LMW-E overexpression causes mitotic defects, chromosome missegregation during metaphase, and anaphase bridges during anaphase, most of which are not detected on EL induction. LMW-E induces additional mitotic defects in cooperation with p53 loss in both normal and tumor cells. Fourth, LMW-E–overexpressing cells fail to arrest in the presence of nocodazole. Collectively, the mitotic defects mediated by LMW-E induction led to failed cytokinesis and polyploidy, suggesting that LMW-E expression primes cells to accrue chromosomal instability by shortening the length of mitosis. Lastly, LMW-E expression in human breast cancer tissues correlates with centrosome amplification and higher nuclear grade. These results suggest that LMW-E overexpression leads to higher centrosome numbers in breast cancer, which is a prerequisite for genomic instability. Cancer Res; 70(12); 5074–84. ©2010 AACR.

Supplementary Movie 6 from Low Molecular Weight Cyclin E Overexpression Shortens Mitosis, Leading to Chromosome Missegregation and Centrosome Amplification

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. [Cancer Res 2007;67(15):7062–7]

Supplementary Figure 1 from Cyclin E Deregulation Impairs Mitotic Progression through Premature Activation of Cdc25C

Supplementary Movie 4 from Low Molecular Weight Cyclin E Overexpression Shortens Mitosis, Leading to Chromosome Missegregation and Centrosome Amplification

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.

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.

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-kβ,; 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.

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.

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.

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.

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-kβ,; 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.

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.

Medullary thyroid cancer (MTC) is an aggressive tumor with frequent lymph node and distant metastasis at diagnosis. Activating mutations of the RET receptor tyrosine kinase occur frequently and prevent apoptosis through inhibition of ATF4, a key transcriptional regulator of endoplasmic reticulum stress. Prior studies have demonstrated shRNA depletion of ATF4 increases RET protein levels and cause resistance to tyrosine kinase inhibitor-mediated cell death. Conversely, forced expression of ATF4 in MTC cells promotes RET ubiquitination and degradation. Here, we report the anti-cancer efficacy of ONC201 in MTC models. ONC201 is a selective DRD2 antagonist that is in clinical trials for a range of advanced cancers. Downstream signaling studies have found that ONC201 induces ATF expression and activates the integrated stress response (ISR) in a variety of cancers. ONC201 decreased cell survival of MTC cell lines (TT and MZCRC1) harboring activating RET mutations and increased the percentage of sub-G1 DNA content. Moreover, ONC201 decreased, in a dose-dependent manner, the abundance of RET protein and induced the mRNA and protein levels of ATF4 and its target genes. In our attempt to uncover RET targets, we performed reverse phase protein array analysis using TT- shRNA-RET knockdown cells. Among the top 5 proteins regulated by RET in this screen, we found insulin-like growth factor-binding protein IGFBP2, to be downregulated in shRNA-RET cells. ELISA based cytokine array of MTC-conditioned media showed high levels of secreted IGFBP2. IGFBP2 have been reported to be an oncogene in many human cancers and we show that elevated IGFBP2 protein levels are associated with poor prognosis of primary MTC. The treatment of MTC cells with ONC201 or overexpression of ATF4 inhibited the secretion of IGFBP2 into the media and decreased IGFBP2 protein levels, suggesting that ONC201 targets IGF signaling through activation of ISR. To investigate the efficacy of ONC201 in vivo, established MTC xenografts (TT or MZCRC1) were treated with a weekly dose of 120 mg/kg orally for 8 weeks and tumor burden was assessed every week. We observed that ONC201 decreased tumor growth of TT and MZCRC1 xenografts by 80-90%. There was no evidence of toxicity, as indicated by weight loss in mice. Immunohistochemical and western blot analysis of tumors at the termination of the study demonstrated decreased IGFBP2 protein levels in mice treated with ONC201. These studies identify IGFBP2 as a novel target of the RET proto-oncogene and demonstrate that ONC201, through its effect on ATF4, reverses RET-mediated signaling. These results support the further investigation of ONC201 as a therapeutic agent for the treatment of MTC alone or in combination with tyrosine kinase inhibitors. Citation Format: Rozita Bagheri-Yarmand, Ling Li, Rohinton Tarapore, Joshua E. Allen, Steven I. Sherman, Robert F. Gagel. ONC201 inhibits RET and IGFBP2 signaling through ATF4 mediated- Integrated stress response in medullary thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1286.

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.

Multiple endocrine neoplasia type 2 (MEN2 ) is a rare disorder with distinct clinical features that were key to the elucidation of its underlying molecular cause. Oncogenic transformation of the thyroid C-cell in MEN2 is highly penetrant and progresses through a hyperplastic process prior to malignancy. Tumor burden is assessed by the measurement of serum calcitonin levels, and thyroidectomy is the primary treatment. Clearly defined kindreds with MEN2 were used to map the causative gene to chromosome 10 and to identify mutations in the RET proto-oncogene. The discovery that MEN2 is caused by activating mutations of the RET gene has served to guide an evolving strategy of prophylactic thyroidectomy for the treatment of hereditary MTC and therapeutic strategies for sporadic MTC, where RET activation in the most commonly observed genetic defect. Thyroid C cells are known to express RET at high levels relative to most other cell types; therefore, aberrant activation of the receptor is thought to preferentially sensitize these cells to transformation. A role of RET in medullary thyroid carcinoma has also served to highlight the importance of the MAPK and PI3K signaling pathways in its initiation and progression. Newly published studies demonstrate equally important functions of RET to prevent apoptosis, perhaps allowing the survival of tumor-initiating cell populations. A growing understanding of how RET interacts with growth and death pathways in normal C-cell function, during oncogenic transformation, and finally in tumor progression has helped drive the development of molecular-targeted therapies for the treatment of metastatic medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is a neuroendocrine malignancy, caused by activating mutations of the RET proto-oncogene, with frequent lymph node and distant metastasis at diagnosis. Tyrosine kinase inhibitors (TKIs) that target the RET have proven effective for treatment of MTC, but resistance to TKIs evolves in half of the treated patients. We have previously shown that the expression levels of Activating Transcription Factor 4 (ATF4) which is a stress-induced transcription factor, significantly decreased or lost in half of the MTC tumors. We also demonstrated that forced expression of ATF4 or TKIs-induced level of ATF4 decreases survival of MTC cells by promoting degradation of RET, blocking the activation of RET downstream signaling pathways and subsequently induces apoptosis. More importantly, ATF4 knockdown by shRNA decreased the sensitivity to tyrosine kinase inhibitor-induced apoptosis. Further thyroid gland of Atf4-knockout mice showed C-cell hyperplasia, a precancerous lesion for MTC suggesting a potential tumor suppressor role of ATF4 in MTC. Stress-induced by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a feature of specialized secretory cells, including MTC cells that secrete calcitonin. ATF4 promotes the induction of apoptosis under persistent stress conditions, although the mechanism is not clearly understood. We hypothesize that the combination of ATF4 inducer and TKIs causes an excessive cellular oxidative stress resulting in an activation of apoptosis, all of which may prevent resistance to TKIs. Here, we show that the ER-associated protein degradation (ERAD) inhibitor, eeyarestatin sensitizes MTC cells to the TKIs including sunitinib and vandetanib. The combination of eeyarestatin and TKIs causes a synergistic induction of ATF4 expression and its target genes, an accumulation of reactive oxygen species and subsequent cell death. Chromatin immunoprecipitation followed by sequencing assay (ChIP-seq) in MTC cells treated with eeyarestatin and immunoprecipitated with ATF4 and acetylated lysine 9 of histone 3, identified transcription Kruppel-like factor 9 (KLF9) gene as a transcriptional target of ATF4 activation. KLF9 plays a functional role in oxidative stress-induced cell death. Treatment with eeyarestatin and vandetanib alone or in combination increases the occupancy of ATF4 at the promoter of KLF9 gene and stimulates the expression of KLF9. Depletion of ATF4 by shRNA leads to downregulation of KLF9 expression and prevents oxidative stress-induced cell death. These findings suggest that induction of the ATF4/KLF9 axis causes oxidative stress leading to excessive ER stress, and subsequent cell death. Thus, combining TKIs and ERAD inhibitors that promote ATF4 levels could be effective therapeutic strategies for treating MTC and preventing resistance to TKIs. Citation Format: Rozita Bagheri-Yarmand, Krishna M. Sinha, Ling Li, Yue Lu, Robert F. Gagel. Tyrosine kinase and ERAD inhibitors promote oxidative stress-induced apoptosis through activation of ATF4 / KLF9 axis in medullary thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2426.

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 G2-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 Ser214, 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 G2-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. Cancer Res; 70(12); 5085–95. ©2010 AACR.

Overexpression of the low molecular weight isoforms (LMW-E) of cyclin E induces chromosome instability; however, the degree to which these tumor-specific forms cause genomic instability differs from that of full-length cyclin E (EL), and the underlying mechanism(s) has yet to be elucidated. Here, we show that EL and LMW-E overexpression impairs the G2-M transition differently and leads to different degrees of chromosome instability in a breast cancer model system. First, the most significant difference is that EL overexpression prolongs cell cycle arrest in prometaphase, whereas LMW-E overexpression reduces the length of mitosis and accelerates mitotic exit. Second, LMW-E–overexpressing cells are binucleated or multinucleated with amplified centrosomes, whereas EL-overexpressing cells have the normal complement of centrosomes. Third, LMW-E overexpression causes mitotic defects, chromosome missegregation during metaphase, and anaphase bridges during anaphase, most of which are not detected on EL induction. LMW-E induces additional mitotic defects in cooperation with p53 loss in both normal and tumor cells. Fourth, LMW-E–overexpressing cells fail to arrest in the presence of nocodazole. Collectively, the mitotic defects mediated by LMW-E induction led to failed cytokinesis and polyploidy, suggesting that LMW-E expression primes cells to accrue chromosomal instability by shortening the length of mitosis. Lastly, LMW-E expression in human breast cancer tissues correlates with centrosome amplification and higher nuclear grade. These results suggest that LMW-E overexpression leads to higher centrosome numbers in breast cancer, which is a prerequisite for genomic instability. Cancer Res; 70(12); 5074–84. ©2010 AACR.

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. [Cancer Res 2007;67(15):7062–7]

Mammalian LIM kinase 1 (LIMK1) phosphorylates and inactivates the actin-binding and -depolymerizing factor cofilin and induces actin cytoskeletal changes. LIMK1 is reported to play an important role in cell motility, but the mechanism of induction of cell motility and the role of LIMK1 in tumor growth, angiogenesis and invasion are poorly understood. Here we show that expression of LIMK1 in MDA-MB-435 human breast cancer cells enhanced cell proliferation and cell invasiveness and promoted in vitro angiogenesis. Since tumor metastasis requires degradation of the extracellular matrix by the serine protease urokinase type plasminogen activator (uPA), we examined the role of LIMK1 in the regulation of uPA/uPAR system. LIMK1 overexpression in breast cancer cells upregulated the uPA system, increased uPA promoter activity, induced uPA and uPAR mRNA and protein expression and induced uPA secretion. In contrast, cells transfected with the catalytically inactive LIMK mutant D460N-LIMK1 did not exhibit these phenotypic changes. Blocking antibodies against uPA and uPAR suppressed LIMK1-induced cell invasiveness. In addition, LIMK1 overexpression increased tumor growth in female athymic nude mice, promoted tumor angiogenesis and induced metastasis to livers and lungs, possibly by increasing uPA expression in the tumors. Finally, LIMK1 and uPAR were coordinately overexpressed in human breast tumors. These results suggested an important role for LIMK1 signaling in breast cancer tumor growth, angiogenesis and invasion and a regulatory connection between LIMK1 and the uPA system.

Emerging data suggest that metastasis-associated protein 1 (MTA1) represses ligand-dependent transactivation functions of estrogen receptor-alpha in cultured breast cancer cells and that MTA1 is upregulated in human breast tumors. However, the role of MTA1 in tumorigenesis in a physiologically relevant animal system remains unknown. To reveal the role of MTA1 in mammary gland development, transgenic mice expressing MTA1 under the control of the mouse mammary tumor virus promoter long terminal repeat were generated. Unexpectedly, we found that mammary glands of these virgin transgenic mice exhibited extensive side branching and precocious differentiation because of increased proliferation of ductal and alveolar epithelial cells. Mammary glands of virgin transgenic mice resemble those from wild-type mice in mid-pregnancy and inappropriately express β-casein, cyclin D1 andβ-catenin protein. Increased ductal growth was also observed in the glands of ovariectomized female mice, as well as of transgenic male mice. MTA1 dysregulation in mammary epithelium and cancer cells triggered downregulation of the progesterone receptor-B isoform and upregulation of the progesterone receptor-A isoform, resulting in an imbalance in the native ratio of progesterone receptor A and B isoforms. MTA1 transgene also increased the expression of progesterone receptor-A target genes Bcl-XL(Bcl2l1) and cyclin D1 in mammary gland of virgin mice, and,subsequently, produced a delayed involution. Remarkably, 30% of MTA1 transgenic females developed focal hyperplastic nodules, and about 7%exhibited mammary tumors within 18 months. These studies establish, for the first time, a potential role of MTA1 in mammary gland development and tumorigenesis. The underlying mechanism involves the upregulation of progesterone receptor A and its targets, Bcl-XL and cyclin D1.

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.

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 β-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 β-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 β-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 β-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.

The basic leucine zipper containing activating transcription factors (ATFs) modulates the expression of growth-regulating genes. In this study, we sought to determine specifically the consequences of ATF4 expression on mammary gland development in transgenic mice. Overexpression of ATF4 severely impaired normal development of the mammary gland, which was associated with reduced proliferation and differentiation of mammary alveolar epithelium and up-regulation of p21(WAF1) and p27(Kip1). In addition, there was also impaired lactation accompanied by decreased expression of alpha-lactoalbumin, whey acidic protein, and beta-casein, possibly because of the down-regulation of STAT5a tyrosine phosphorylation. Mammary gland involution in ATF4-transgenic mice was accelerated, compared with wild type littermates by whole mount analysis. In addition, day 18 of lactation in transgenic mice was phenotypically equivalent to day 3 of involution in wild type mice, as determined by the TUNEL assay and expression of Bax. The concentration of the proapoptotic molecule caspase-3 was increased during lactation in ATF4-transgenic animal. Mammary glands from ATF4-transgenic mice also showed significant nuclear translocation of activated STAT3 and up-regulation of one of its target genes, insulin-like growth factor-binding protein-5, which is thought to facilitate apoptosis by sequestering insulin-like growth factor. Together, these findings suggest that ATF4 may play a role during mammary gland development and that down-regulation of ATF4 may be important for the onset of involution in the mammary gland.

Oestrogen receptor (ER) is a good prognostic marker for the treatment of breast cancers. Upregulation of metastatic tumour antigen 1 (MTA1) is associated with the invasiveness and metastatic potential of several human cancers and acts as a co-repressor of nuclear ER-alpha. Here we identify a naturally occurring short form of MTA1 (MTA1s) that contains a previously unknown sequence of 33 amino acids with an ER-binding motif, Leu-Arg-Ile-Leu-Leu (LRILL). MTA1s localizes in the cytoplasm, sequesters ER in the cytoplasm, and enhances non-genomic responses of ER. Deleting the LRILL motif in MTA1s abolishes its co-repressor function and its interaction with ER, and restores nuclear localization of ER. Dysregulation of human epidermal growth factor receptor-2 in breast cancer cells enhances the expression of MTA1s and the cytoplasmic sequestration of ER. Expression of MTA1s in breast cancer cells prevents ligand-induced nuclear translocation of ER and stimulates malignant phenotypes. MTA1s expression is increased in human breast tumours with no or low nuclear ER. The regulation of the cellular localization of ER by MTA1s represents a mechanism for redirecting nuclear receptor signalling by nuclear exclusion.

The activating transcription factor 4 (ATF4) plays a central role in activation of the integrated stress response pathway. Targeted deletion of Atf4 in mouse thyroid gland causes C-cell hyperplasia, a precancerous lesion for medullary thyroid cancer (MTC). Activating mutations of the tyrosine kinase RET are associated with oncogenic function in MTC progression. We have reported that RET is a serine-threonine dual specificity kinase, phosphorylates ATF4, and inhibits expression of the ATF4 target proapoptotic genes. Forced ATF4 expression in MTC cells decreased their survival through ubiquitin-mediated degradation of RET protein levels. We hypothesize that inactivation of ATF4 by RET is a central impediment to induction of apoptosis in response to TKI therapy. Further upregulation of ATF4 would render tumor cells exquisitely sensitive to stress induced apoptosis. Here, we show that the treatment of MTC cells with tyrosine kinase inhibitors upregulates ATF4 expression and induces Endoplasmic reticulum (ER) stress. Eeyarestatin is an ER-associated protein degradation (ERAD) inhibitor that elicit an integrated stress response program at the ER, which results in activation of ATF4 to stimulate expression of the apoptotic genes, PMAIP1 and BBC3, resulting into apoptotic cell death. We found that treatment of MTC cell lines, TT and MZCRC1 cells, with eeyarestatin not only induces ATF4 but also downregulates RET protein levels. The combinations of TKIs (vandetanib, sunitinib, cabozantinib) with eeyarestatin promote apoptotic cell death in a synergistic manner shown by cleaved PARP/ cleaved casapase3 staining using flow cytometry. Western blot analysis showed that combination therapy increased ATF4 levels and its targets, NOXA and PUMA levels as compared to each drug treatment alone. We applied genome-wide Chromatin Immunoprecipation Sequencing (ChIP-seq) to identify genes directly regulated by ATF4 in eeyarestatin-treated MZCRC1 cells. ChIP-seq analysis identified many previously known and unknown ATF4 target genes. We have identified the stress response, proapoptotic genes, inhibitors of MAPK, mTOR and βcatenin-wnt pathways with tumor suppressor function, including MKNK2, TCF3, APC2, KLF9, LZTFL1, SIAH1. Gene expression analysis was performed to further validate the activation of those genes in ATF4-overexpressing and eeyarestatin-treated cells. We found that combination of TKI and eeyarestatin synergistically upregulated expression of ATF4, and its targets APC2, LZTFL1, BBC3, MKNK2, and SIAH1. These results suggest that induction of ATF4 in combination with TKIs could be a promising therapeutic strategy to treat patients with medullary thyroid cancer. Citation Format: Rozita Bagheri-Yarmand, Krishna Sinha, Yue Lu, Robert Gagel. Tyrosine kinase and ERAD inhibitors synergize to promote apoptosis through ATF4 induction in medullary thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-112. doi:10.1158/1538-7445.AM2017-LB-112

We have previously shown that carboxymethyl dextran benzylamide (CMDB7), a heparin-like molecule, inhibits the growth of tumors xenografted in nude mice, angiogenesis, and metastasis by altering the binding of angiogenic growth factors, including platelet-derived growth factor, transforming growth factor beta, and fibroblast growth factor 2, to their specific receptors. In this study, we explore the effect of CMDB7 on the most specific angiogenic growth factor, vascular endothelial growth factor 165 (VEGF(165)). We demonstrate here that CMDB7 inhibits the mitogenic effect of VEGF(165) on human umbilical vein endothelial cells (HUV-ECs) by preventing the VEGF(165)-induced VEGF receptor-2 (KDR) autophosphorylation and consequently a specific intracellular signaling. In competition experiments, the binding of (125)I-VEGF(165) to HUV-ECs is inhibited by CMDB7 with an IC(50) of 2 microm. Accordingly, CMDB7 inhibits the cross-linking of (125)I-VEGF(165) to the surface of HUV-ECs, causing the disappearance of both labeled complexes, 170-180 and 240-250 kDa. We show that CMDB7 increases the electrophoretic mobility of VEGF(165), thus evidencing formation of a stable complex with this factor. Moreover, CMDB7 reduces the (125)I-VEGF(165) binding to coated heparin-albumin and prevents a heparin-induced increase in iodinated VEGF(165) binding to soluble (125)I-KDR-Fc chimera. Concerning KDR, CMDB7 has no effect on (125)I-KDR-Fc electrophoretic migration and does not affect labeled KDR-Fc binding to coated heparin-albumin. In the presence of VEGF(165), (125)I-KDR-Fc binding to heparin is enhanced, and under these conditions, CMDB7 interferes with KDR binding. These data indicate that CMDB7 effectively inhibits the VEGF(165) activities by interfering with heparin binding to VEGF(165) and VEGF(165).KDR complexes but not by direct interactions with KDR.

Etk/Bmx, a member of the Tec family of nonreceptor protein-tyrosine kinases, is characterized by an N-terminal pleckstrin homology domain and has been shown to be a downstream effector of phosphatidylinositol 3-kinase. P21-activated kinase 1 (Pak1), another well characterized effector of phosphatidylinositol 3-kinase, has been implicated in the progression of breast cancer cells. In this study, we characterized the role of Etk in mammary development and tumorigenesis and explored the functional interactions between Etk and Pak1. We report that Etk expression is developmentally regulated in the mammary gland. Using transient transfection, coimmunoprecipitation and glutathione S-transferase-pull down assays, we showed that Etk directly associates with Pak1 via its N-terminal pleckstrin homology domain and also phosphorylates Pak1 on tyrosine residues. The expression of wild-type Etk in a non-invasive human breast cancer MCF-7 cells significantly increased proliferation and anchorage-independent growth of epithelial cancer cells. Conversely, expression of kinase-inactive mutant Etk-KQ suppressed the proliferation, anchorage-independent growth, and tumorigenicity of human breast cancer MDA-MB435 cells. These results indicate that Pak1 is a target of Etk and that Etk controls the proliferation as well as the anchorage-independent and tumorigenic growth of mammary epithelial cancer cells.

Epidermal growth factor (EGF) family of growth factors and their receptors regulate normal and cancerous epithelial cell proliferation, a process that can be suppressed by antireceptor blocking antibodies. To identify genes whose expression may be modulated by antireceptor blocking antibodies, we performed a differential display screen with cells grown in the presence or absence of antireceptor blocking antibodies; isolates from one cDNA clone were 100% identical to human heterogeneous nuclear ribonucleoprotein K (hnRNP K), a protein with a conserved KH motif and RGG boxes, has been implicated in such functions as sequence-specific DNA binding, transcription, RNA binding, and nucleocytoplasmic shuttling. Both EGF and heregulin-beta1 induced expression of hnRNP K mRNA and protein in human breast cancer cells. This growth factor-mediated hnRNP K expression was effectively blocked by pretreatment of cultures with humanized anti-EGF receptor (EGFR) antibody C225, or anti-human epidermal growth factor receptor-2 (HER2) antibody. Anti-EGFR monoclonal antibody also caused regression of human tumor xenografts and reduction in hnRNP K levels in athymic mice. Samples from grade III human breast cancer contained more hnRNP K protein than samples from grade II cancer. Finally, overexpression of hnRNP K in breast cancer cells significantly increased target c-myc promoter activity and c-Myc protein, hnRNP K protein levels, and enhanced breast cancer cell proliferation and growth in an anchorage-independent manner. These results suggested that the activity of human EGF receptor family members regulates hnRNP K expression by extracellular growth promoting signals and that therapeutic humanized antibodies against EGFR and HER2 can effectively block this function.

Activation of the heregulin/HER2 pathway in oestrogen receptor (ER)-positive breast-cancer cells leads to suppression of oestrogen-receptor element (ERE)-driven transcription and disruption of oestradiol responsiveness, and thus contributes to progression of tumours to more invasive phenotypes. Here we report the identification of metastatic-associated protein 1 (MTA1), a component of histone deacetylase (HDAC) and nucleosome-remodelling complexes, as a gene product induced by heregulin-beta1 (HRG). Stimulation of cells with HRG is accompanied by suppression of histone acetylation and enhancement of deacetylase activity. MTA1 is also a potent corepressor of ERE transcription, as it blocks the ability of oestradiol to stimulate ER-mediated transcription. The histone-deacetylase inhibitor trichostatin A blocks MTA1-mediated repression of ERE transcription. Furthermore, MTA1 directly interacts with histone deacetylase-1 and -2 and with the activation domain of ER-alpha. Overexpression of MTA1 in breast-cancer cells is accompanied by enhancement of the ability of cells to invade and to grow in an anchorage-independent manner. HRG also promotes interaction of MTA1 with endogenous ER and association of MTA1 or HDAC with ERE-responsive target-gene promoters in vivo. These results identify ER-mediated transcription as a nuclear target of MTA1 and indicate that HDAC complexes associated with the MTA1 corepressor may mediate ER transcriptional repression by HRG.

Heregulin-beta1 promotes the activation of p21-activated kinase 1 (Pak1) and the motility and invasiveness of breast cancer cells. In this study, we identified vascular endothelial growth factor (VEGF) as a gene product induced by heregulin-beta1. The stimulation by heregulin-beta1 of breast cancer epithelial cells induced the expression of the VEGF mRNA and protein and its promoter activity. Heregulin-beta1 also stimulated angiogenesis in a VEGF-dependent manner. Herceptin, an anti-HER2 antibody inhibited heregulin-beta1-mediated stimulation of both VEGF expression in epithelial cells and angiogenesis in endothelial cells. Because the activation of Pak1 and VEGF expression are positively regulated by heregulin-beta1, we hypothesized that Pak1 regulates VEGF expression, and hence explored the role of Pak1 in angiogenesis. We provide new evidence to implicate Pak1 signaling in VEGF expression. Overexpression of a kinase-dead K299R Pak1 leads to suppression of VEGF promoter activity, as well as VEGF mRNA expression and secretion of VEGF protein. Conversely, kinase-active T423E Pak1 promotes the expression and secretion of VEGF. Furthermore, expression of the heregulin-beta1 transgene, HRG, in harderian tumors in mice enhances the activation of Pak1 as well as expression of VEGF and angiogenic marker CD34 antigen. These results suggest that heregulin-beta1 regulates angiogenesis via up-regulation of VEGF expression and that Pak1 plays an important role in controlling VEGF expression and, consequently, VEGF secretion and function.