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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.

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.

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.

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.

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.

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

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.

Medullary thyroid carcinoma (MTC) is derived from the calcitonin-producing C-cells of the thyroid gland. Oncogenic mutations of the RET proto-oncogene are found in all hereditary MTC and ∼45% of the sporadic cases. However, it is clear that additional genetic lesions contribute to the development of both RET-dependent and RET independent MTC. We analyzed 30 primary medullary thyroid cancer (MTC) tumors by comparative genomic hybridization (CGH) arrays (Agilent 244K) and a subset of those tumors by ultra-high-resolution SNP arrays (Affymetrix SNP6.0). A consistent finding with both arrays was a loss localized to 22q13.1 in 40% of tumors. This region contains the ATF4 gene and loss of this gene was validated by real time PCR. ATF4 is a central mediator of integrated stress response pathway and activates expression of a cohort of downstream target genes known to be involved in cell survival, apoptosis, autophagy and senescence. We hypothesize that the loss of ATF4 expression contributes to MTC progression through mechanisms involving aberrant proliferation and escape from apoptosis. We present several findings to support this. First, ATF4 protein levels were found downregulated in 50% MTC tumor tissues (n = 43) and low ATF4 expression was associated with poor overall survival in MTC patients (HR = 5.015, 95% CI: 1.38-10.64), P = 0.013). A negative correlation was observed between RET and ATF4 protein expression in MTC tumors (r = -0.89, R square = 0.799, p Citation Format: Rozita Bagheri-Yarmand, Gilbert J. Cote, Elizabeth G. Grubbs, Michelle D. Williams, Steven I. Sherman, Robert F. Gagel. ATF4 is a putative tumor suppressor gene in medullary thyroid cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3660.

Androgen ablation by pharmacological use or castration is a standard therapy for treating prostate cancer (PCa), however, at the later stage PCa becomes castration-resistant and is metastasized primarily to bones. Epigenetic changes that cause dysregulated gene expression during progression and metastasis of androgen-independent prostate cancer are not clearly understood. We previously reported that the jumonji C domain-containing NO66 is a histone demethylase specific for H3K4me3 and H3K36me3, which negatively regulates osteoblast differentiation and bone formation. By analyzing a multi-cancer tissue microarray, the serendipitous observation was made that NO66 is overexpressed in a variety of cancers. Our objective was to examine NO66's role in the pathogenesis of prostate cancer and bone metastasis. Our findings indicate that NO66 knockdown resulted in significant decreases in the proliferation and invasiveness of prostate cancer cells (PC3 and DU145) while overexpressing NO66 promoted their proliferation and invasion. Furthermore, NO66 depletion resulted in a two- to three fold decrease in colony formation and anchorage-independent growth of PCa cells suggesting an oncogenic function of NO66. RNAseq analysis comparing between control and NO66-depleted DU-145 cells revealed a repertoire of differentially expressed genes. NO66 up-regulates genes with proliferative activity such as chemokine CXCL5, growth factor IGFBP5, IL6, WNT and MAPK4 pathway, which are known to drive PCa proliferation. Furthermore, ChIP sequencing for NO66 and H3K9AC in NO66-overexpressing PC3 cells revealed that interaction sites of NO66 overlap with peaks of H3K9AC in majority of the genes. These data clearly indicate that NO66 activates a cohort of genes which are involved in tumor progression and metastasis. Strikingly, implanting NO66-overexpressing prostate cancer cells into the femur of SCID male mice caused massive femoral bone loss after three weeks, which suggests that NO66 causes osteolytic bone lesions similar to the clinico-pathological features of advanced PCa. Finally, analysis of tumor xenografts derived from patients by immunohistochemistry showed high expression of NO66 in high grade advanced PCa compared to low grade and hyperplasia suggesting that NO66 may be used as a biomarker to distinguish aggressive prostate cancer from indolent stage. Our data suggest that NO66 plays an important role in cell proliferation, cell invasion and bone metastasis of prostate cancer. Citation Format: Rozita Bagheri-Yarmand, Nora Navone, Xinhai Wan, Christopher Logothetis, Johnny Huard, Krishna Sinha. Oncogenic and osteolytic function of histone demethylase NO66 in castration resistant prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 693.

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]

RET proto-oncogene, a receptor tyrosine kinase (RTK), activates a complex network of signal transduction pathways that when unregulated contributes to cell transformation. Germ line missense mutations, which give rise to constitutively active oncogenic RET, were found to cause multiple endocrine neoplasia type 2 (MEN2), a dominant inherited cancer syndrome that affects multiple neuroendocrine organs. Recent findings also suggest that impairment of RTK down-regulation or deactivation may be involved in tumorigenesis. The molecular mechanism that functions to attenuate RET signaling remain elusive. ATF4 is a central mediator of integrated stress response pathway. Activation of ATF4 results in tumor cell death under stress conditions thereby suggesting that ATF4 agonists constitute a potential therapeutic strategy for inhibiting tumor growth. Here, we report that ATF4 overexpression in medullary thyroid cancer cells harboring RET C634W mutation decreases RET protein expression but not the mRNA levels. In addition, ATF4 accelerates RET turnover and promotes its ubiquitination. Moreover, a RET kinase dead mutant is more stable than wild type or M918T RET suggesting that kinase activity is required for ATF4-mediated degradation. Finally, ATF4 depletion upregulates RET protein levels and RET autophosphorylation. Our findings unravel ATF4 as a feedback negative attenuator of RET signaling that could be used as a potential therapeutic target for medullary thyroid cancer or other malignancy with RET abnormalities. Citation Format: Rozita Bagheri-Yarmand, Gilbert J. Cote, Robert F. Gagel. Activating transcription factor 4 regulates RET autophosphorylation and ubiquitin-dependent degradation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5262. doi:10.1158/1538-7445.AM2014-5262