Decreased Anchorage Independent Growth Research Articles

Chronic nickel exposure alters extracellular vesicles to mediate cancer progression via sustained NUPR1 expression

Cancer cells release extracellular vesicles (EVs) that participate in altering the proximal tumor environment and distal tissues to promote cancer progression. Chronic exposure to nickel (Ni), a human group I carcinogen, results in epigenetic changes that promotes epithelial to mesenchymal transition (EMT). Cells that undergo EMT demonstrate various molecular changes, including elevated levels of the mesenchymal cadherin N-cadherin (N-CAD) and the transcription factor Zinc finger E-box binding homeobox 1 (ZEB1). Moreover, the molecular changes following EMT induce changes in cellular behavior, including anchorage-independent growth, which contributes to cancer cells detaching from tumor bulk during the metastatic process. Here, we present data demonstrating that EVs from Ni-exposed cells induce EMT in recipient BEAS-2B cells in the absence of Ni. Moreover, we show evidence that the EVs from Ni-altered cells package the transcription factor nuclear protein 1 (NUPR1), a transcription factor associated with Ni exposure and cancer progression. Moreover, our data demonstrates that the NUPR1 in the EVs becomes part of the recipient cell proteomic milieu and carry the NUPR1 to the nuclear space of the recipient cell. Interestingly, knockdown of NUPR1 in Ni-transformed cells suppressed NUPR1 packaging in the EVs, and nanoparticle tracking analysis (NTA) demonstrated decreased EV release. Reduction of NUPR1 in EVs resulted in diminished EMT capacity that resulted in decreased anchorage independent growth. This study is the first to demonstrate the role of NUPR1 in extracellular vesicle-mediate cancer progression.

Abstract 669: Nuclear FAK drives thyroid cancer growth and survival

Abstract Background: Late stage thyroid cancers characterized by metastasis and extranodal invasion have a poor prognosis compared to those with localized disease. However, there are limited therapeutic options and few biomarkers to indicate which patients will develop aggressive disease. Our lab has identified Focal Adhesion Kinase (FAK) as a key regulator of thyroid cancer growth, invasion, and metastasis. FAK is a non-receptor tyrosine kinase that is auto-phosphorylated at tyrosine 397 (Y397) in response to integrin or growth factor receptor signaling resulting in the activation of downstream signaling pathways. While FAK is predominantly localized at the plasma membrane, FAK has also been shown to accumulate in the nucleus via a nuclear localization sequence (NLS) to promote cell survival. We have found that FAK localizes to the nucleus in a subset of thyroid cancer patient tumors and that phosphorylated Y397 FAK (pY397 FAK) specifically accumulates in the nucleolus. The nucleolus plays a key role in cancer progression through the synthesis of ribosomal RNA (rRNA) and subsequent increase in ribosome biogenesis, protein synthesis, and tumor growth. It is unclear how pY397 FAK localizes to the nucleolus and what the function of pY397 FAK is in the nucleolus. Hypothesis: Nuclear FAK drives thyroid cancer growth and survival through the phosphorylation of nucleolar proteins involved in rRNA transcription. Results: To address the functional role of FAK in the nucleus, we excluded FAK from the nucleus by mutating its NLS and found that it significantly decreased anchorage independent growth compared to wild type (WT) FAK in our thyroid cancer cells (BCPAP and 8505C). Importantly, non-phosphorylatable FAK mutant (Y397F) and kinase dead FAK mutant also decreased growth indicating that nuclear and pY397 FAK are required for anchorage independent growth. Furthermore, we found that forcing FAK into the nucleus with an SV40 NLS resulted in increased FAK nucleolar accumulation which was eliminated when FAK is forced into the nucleus with Y397F FAK. These data indicate that pY397 FAK is required for FAK nucleolar accumulation. To investigate the role of pY397 FAK in the nucleolus, we performed BioID to identify novel protein-protein interactions for WT FAK and Y397F FAK. We found that pY397 FAK interacts with a network of nucleolar proteins including NPM1, TOP1, and DDX46. Interestingly, the majority of these nucleolar proteins are involved in transcription of pre-rRNA which is essential for protein synthesis and growth. Of note, Nucleophosmin 1 (NPM1) is an endoribonuclease that regulates pre-rRNA synthesis through the cleavage of the 46S rRNA transcript. We confirmed that pY397 FAK specifically interacts with NPM1 by Proximity Ligation Assay. Conclusion: These data indicate that pY397 FAK interacts with a network of nucleolar proteins involved in rRNA transcription and that nuclear pY397 FAK drives growth and survival in thyroid cancer. Citation Format: Meghan Kellett, Vibha Sharma, Brittelle Kessler, Sharon Sams, Molishree Joshi, Rebecca E. Schweppe. Nuclear FAK drives thyroid cancer growth and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 669.

Novel Role of Nitric‐Oxide Synthase Trafficking Inducer (NOSTRIN) in Combating Colon Cancer Aggressiveness

Colorectal cancer (CRC) is a global health problem, with approximately 1.2 million new cases per year. CRC is the third most common cancer in men (10% of all cancer cases) and the second most common in women (9.4% of all cancer cases) making it the fourth highest cause of cancer death worldwide. Being a highly heterogeneous and aggressive disease, rate of its early detection is very less and incidence of rapid malignant proliferation is high. Further identification of new targets to restrict CRC progression is an absolute need of the hour. Interestingly, a novel molecule, NOSTRIN, shown to be anti-angiogenic, anti-invasive and anti-inflammatory, is abundantly expressed in colon and according to the Human Protein Atlas its expression is also detected in colorectal cancer samples both in RNA and protein levels. Furthermore, NOSTRIN has been demonstrated as a negative regulator of disease aggressiveness in pancreatic cancer patients and its increased expression was found to be associated with increased survival of patients. However, so far there has been no report directly linking NOSTRIN with colorectal cancer. We hypothesized that NOSTRIN plays a significant role in regulating a) colon cancer aggressiveness & b) cancer cell stemness. In our study, NOSTRIN expression was found to be inversely related to increased aggressiveness of colon cancer cell lines both in transcript and protein levels. Stable ectopic overexpression of NOSTRIN in CRC cell line led to decrease in expression of gene signatures for Epithelial to Mesenchymal Transition (EMT), one of the hallmark events of cancer, as established by our EMT array. This array data was further validated by western blotting. In line with this finding, migration and invasion ability of CRC cells were curbed by NOSTRIN overexpression as demonstrated by scratch wound and cell invasion assay, respectively. Furthermore, NOSTRIN overexpression remarkably decreased anchorage independent growth ability of CRC cells as shown by soft agar colony formation assay. These results indicate NOSTRIN reduces the metastatic potential of CRC cell lines. Interestingly, BrdU incorporation was significantly reduced in CRC cells by presence of excess NOSTRIN. In addition, heightened NOSTRIN expression led to decreased colonosphere formation and reduced expression of stemness markers, such as, CD133, CD44 and EpCAM by CRC cell lines. NOSTRIN's interaction with Cyclin Dependent Kinase1 (CDK1) and increased inhibitory CDK1 phosphorylation (Y15 and T14) in NOSTRIN over-expressing CRC cell lines might contribute to its inhibitory role in CRC cells’ stemness. Finally, a clear negative correlation between NOSTRIN expression and CRC disease progression was demonstrated in human patient samples using CRC cDNA array. Taken together, this report established a significant role of NOSTRIN in inhibiting a) EMT, b) metastatic potential and c) self-renewal of CRC cells.

Abstract P3-05-03: Identification of epigenetically silenced breast cancer driver genes

Abstract Breast cancer is clinically and molecularly complex disease driven by aberrant genetic and epigenetic alterations. Epigenetic alterations in particular DNA methylation changes are one of the most important events involved in breast cancer initiation and progression. Previous reports identified many aberrant DNA methylation signatures associated with molecular subtypes of breast cancer and over 100 candidate genes with promoter hypermethylation in breast cancer. However, it remains elusive which of these genes with promoter hypermethylation play “driver” role in tumorigenesis. In previous studies, the average gain of DNA methylation across all cancer samples compared to the average DNA methylation in normal samples has been the criterion to select for potential targets. However, known tumor suppressor driver genes regulated by methylation are relatively infrequently altered in target cancers. Therefore, we propose the paradoxical hypothesis that identifying hypermethylated cancer drivers require focusing on infrequent rather than frequent events. Hence, to identify these potential driver genes, we developed an algorithm with two unique properties. First, unlike previous studies we focused on targets that gained DNA methylation relatively infrequently (10-40%) and that lost expression in breast cancer. Second, using this algorithm, we distinguished cancer dependent gain of DNA methylation from age-dependent gain of methylation. To discern age dependent and independent DNA methylation changes, we generated DNA methylation sequencing data on 29 normal purified breast epithelium (age range 33-82 years old). Furthermore, to study the biological effects of the overexpression or downregulation of these genes, we generated DNA methylation sequencing data on 6 breast cancer cell lines. We also used DNA methylation and RNA expression datasets (675 cancer, 100 normal) available through the TCGA. Using our algorithm, we identified 53 genes with age independent promoter hypermethylation and loss of expression in TCGA tumor samples. To begin testing the biological effects of these driver genes, we performed canonical pathway enrichment analyses using Ingenuity Pathway Analysis software. We also investigated the mutational status of these genes and their molecular subtype enrichment. Based on these analyses, we picked 12 genes (C10orf125, RUNX3, YOD1, FXYD5, SMOC1, SLC16A5, RNLS, DKK1, PNPLA3, FZD10, RND2, and PLCB1) for further study. We stably overexpressed these potential driver genes in different breast cancer cell lines. Twelve genes out of the 12 tested, slowed cell proliferation and 9 decreased anchorage independent growth. We further validated these driver genes by knocking them out in normal human mammary epithelial cells using CRISPR/Cas9 tool. The loss of these genes, increased cell proliferation rate in normal human mammary epithelial cells compared to the control cells. In conclusion, based on our preliminary data, using bioinformatics tools as well as functional assays, we identified epigenetically altered breast cancer driver genes. Identifying and deciphering true epigenetic cancer drivers could potentially lead to the development of therapeutic drugs targeting these genes and/or targeting pathway dependence. Citation Format: Panjarian S, Madzo J, Slater C, Jelinek J, Chen X, Issa J-P. Identification of epigenetically silenced breast cancer driver genes [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-05-03.

Ewing sarcoma resistance to SP-2509 is not mediated through KDM1A/LSD1 mutation.

Ewing sarcoma is the second most common solid bone malignancy diagnosed in pediatric and young adolescent populations. Despite global co-operative efforts, outcomes for patients with relapsed and refractory disease remains obstinately poor. It has become increasingly clear that disruption of the epigenome as a result of alterations in epigenetic regulators, plays a pivotal role in tumorigenesis. As such, this study investigated Ewing sarcoma mechanisms of acquired resistance to the small molecule reversible lysine specific demethylase (LSD1/KDM1A) inhibitor SP-2509. Surprisingly, whole exome sequencing analysis of our generated A673 SP-2509 drug resistant cell line revealed an absence of mutations in KDM1A. Compared to parental counterparts, SP-2509 drug resistant cells demonstrated decreased anchorage independent growth capacity, enhanced sensitivity to the HDAC inhibitors vorinostat/entinostat and a distinct transcriptional profile that was enriched for extracellular matrix proteins. SP-2509 drug resistant cells also exhibited elevated expression levels of the multi-drug resistance genes ABCB1, ABCC3, and ABBC5 and decreased expression of the transcriptional repressor RCOR1/CoREST. Following several months of SP-2509 withdrawal, low level SP-2509 resistance was still apparent (6.3 fold increase in IC50), with drug resistant cell populations maintaining their distinct transcriptional profile. Furthermore, compared to parental cells, washout drug resistant lines displayed equal sensitivity to the standard Ewing sarcoma chemotherapeutic agent's vincristine and doxorubicin. Together these findings indicate that resistance to SP-2509 is not fully reversible or driven by direct mutation in KDM1A.

Regulation of the oncogenic phenotype by the nuclear body protein ZC3H8

BackgroundThe Zc3h8 gene encodes a protein with three zinc finger motifs in the C-terminal region. The protein has been identified as a component of the Little Elongation Complex, involved in transcription of small nuclear RNAs. ZC3H8 is overexpressed in a number of human and mouse breast cancer cell lines, and elevated mRNA levels are associated with a poorer prognosis for women with breast cancer.MethodsWe used RNA silencing to decrease levels of expression in mouse mammary tumor cells and overexpression of ZC3H8 in cells derived from the normal mouse mammary gland. We measured characteristics of cell behavior in vitro, including proliferation, migration, invasion, growth in soft agar, and spheroid growth. We assessed the ability of these cells to form tumors in syngeneic BALB/c mice. ZC3H8 protein was visualized in cells using confocal microscopy.ResultsTumor cells with lower ZC3H8 expression exhibited decreased proliferation rates, slower migration, reduced ability to invade through a basement membrane, and decreased anchorage independent growth in vitro. Cells with lower ZC3H8 levels formed fewer and smaller tumors in animals. Overexpression of ZC3H8 in non-tumorigenic COMMA-D cells led to an opposite effect. ZC3H8 protein localized to both PML bodies and Cajal bodies within the nucleus. ZC3H8 has a casein kinase 2 (CK2) phosphorylation site near the N-terminus, and a CK2 inhibitor caused the numerous PML bodies and ZC3H8 to coalesce to a few larger bodies. Removal of the inhibitor restored PML bodies to their original state. A mutant ZC3H8 lacking the predicted CK2 phosphorylation site showed localization and numbers of ZC3H8/PML bodies similar to wild type. In contrast, a mutant constructed with a glutamic acid in place of the phosphorylatable threonine showed dramatically increased numbers of smaller nuclear foci.ConclusionsThese experiments demonstrate that Zc3h8 expression contributes to aggressive tumor cell behavior in vitro and in vivo. Our studies show that ZC3H8 integrity is key to maintenance of PML bodies. The work provides a link between the Little Elongation Complex, PML bodies, and the cancer cell phenotype.

Abstract 4380: Role of FAM3B/PANDER in inhibition of cell death and tumor growth in breast and prostate cancer

Abstract FAM3B/PANDER is a novel cytokine-like protein that induces apoptosis in beta-cells and regulates the effects of insulin in peripheral tissues. Since previous data revealed that FAM3B can be expressed by prostate and breast tumors, we evaluated the role of this cytokine in prostate and breast tumor progression. FAM3B expression was compared by quantitative PCR in LnCAP, PC-3 and DU145 prostate tumor cell lines and MCF-7 and MDA-MB- 231 breast tumor cell lines. After treatment with either recombinant FAM3B protein or secreted FAM3B obtained from conditioned media (CM) derived from FAM3B-overexpressing 293T cells, the cell death and viability of DU145 and MDA-MB- 231 cell lines were evaluated. DU145 and MDA-MB- 231 cells overexpressing FAM3B protein were produced by lentiviral-mediated transduction of full-length FAM3B cDNA. Cell viability and apoptosis were analyzed in DU145-FAM3B and MDA-231-FAM3B cells after treatment with cell death inducers. Anchorage-independent growth and scratch wound assays were used to evaluate in vitro tumorigenicity and cell migration, respectively. In vivo tumorigenicity and invasiveness were evaluated by tumor xenograft growth in nude mice. We observed that FAM3B was highly expressed by hormone responsive cells (LnCAP and MCF-7) and low expressed in unresponsive hormone cells (PC-3, DU145 and MDA-MB- 231). Cell viability and survival of DU145 and MDA-MB- 231 cells increased after exogenous treatment with recombinant FAM3B protein or CM containing FAM3B-secreted protein. Overexpression of FAM3B in DU145 and MDA-MB- 231 cells promoted an inhibition of apoptosis triggered by TNF-alpha and staurosporine. Cell death inhibition was accompanied by increased gene expression of the anti-apoptotic Bcl-2 and Bcl-xL genes, and by slight decrease in expression of pro-apoptotic gene Bax and with a decrease of caspases proteolytic activities. When compared to control, cells overexpressing FAM3B displayed a decreased anchorage independent growth and increased motility in vitro, as well as an increased tumor growth in xenografted nude mice. In agreement Immunohistochemistry analysis from tumor xenografts revealed similar anti-apoptotic phenotype in FAM3B overexpression, with an increase of Bcl-2. The results showed that FAM3B was capable to activates pro-survival mechanisms with an increasing of tumor growth by, activation of Bcl-2 and Bcl-xl pathways, suggesting the role of this cytokine in breast and in prostate tumors progression. Citation Format: Izabela D. Caldeira. Role of FAM3B/PANDER in inhibition of cell death and tumor growth in breast and prostate 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 4380.

Abstract 5259: Urolithin A prevents pancreatic tumor growth and increases survival by inhibiting PI3K/PDK1 and STAT3 signaling

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related mortality in the United States. Most patients present with an advanced disease and the majority die within five years, many surviving less than six months. Cytotoxic chemotherapy including Gemcitabine (Gem), FOLFIRINOX, nab-paclitaxel offer modest improvement in survival, albeit at the cost of increased side effects and unwanted toxicities. Therefore, developing novel chemotherapeutic agents for PDAC treatment is critical to improve survival. Ellagic acid/ellatitannins are abundantly present in the pomegranate and berries, are actively metabolized by the intestinal microflora to Urolithin A (UA). Oral administration of UA has shown to be highly bioavailable and non-toxic. UA inhibits multiple kinases that are known to be involved in PDAC progression and metastasis. Therefore, we hypothesized that UA would elicit potent anti-cancer therapeutic potential in PDAC. The effect of UA on kinase activity was assessed . Inhibition of AKT (downstream of PI3K/PDK1), p70 S6 Kinase (PS6K) and STAT3 activation was quantified in PDAC cells treated with UA in dose-dependent manner. The mechanism of action was validated for UA’s activity on PI3K/PDK1, PS6K and STAT3 activation using immunoblot analysis. MiaPaCa2 cells were treated with specific inhibitors for either AKT (MK2206) or STAT3 (AZD1480) and analyzed for tumorigenicity. UA treated PDAC cells were analyzed for cell proliferation, cell invasion and colony formation. Cell cycle analysis and cell apoptosis were measured by flow cytometry. To test the efficacy of UA in vivo, cells were implanted subcutaneously in athymic nude mice. The animals received UA daily and tumor volume was measured for 5 weeks. Next, we assessed tumor growth and overall survival (OS) in PKT (Ptf1acre/+;LSL-KrasG12D;Tgfbr2fl/fl) mice, an aggressive genetically engineered PDAC mouse model, in response to UA and/or Gem treatment. Tissues from the xenografts and PKT mice treated with vehicle or UA were analyzed for cell proliferation (Ki67) and apoptosis (cleaved Caspase 3) by immunohistochemistry. High expression levels of activated STAT3 or AKT correlate with decreased survival in PDAC. UA treated MiaPaCa2 cells showed significant dose-dependent increase in apoptosis and decrease in anchorage-independent growth. UA inhibited AKT, PS6K and STAT3 signaling. As a single agent, UA effectively reduced in vivo PDAC tumor growth. Immunohistochemistry of UA treated tissues from tumor xenografts and PKT mice showed inhibition of Ki67 positive tumor cells and increased cleaved caspase 3 staining. PKT mice treated with UA showed a decrease in tumor size and an increased OS when compared to vehicle or Gem treated mice alone. These findings show that UA is a novel inhibitor/modulator/regulator for multiple signaling pathways in PDAC. These results suggest UA has potential for pre-clinical development in pancreatic cancer. Citation Format: Supriya Srinivasan, Venkatakrishna Jala, Kumar Honnenahally, Jason Castellanos, Praveen Kumar Vermula, Michael VanSaun, Nipun Merchant, Nagaraj Nagathihalli. Urolithin A prevents pancreatic tumor growth and increases survival by inhibiting PI3K/PDK1 and STAT3 signaling [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 5259. doi:10.1158/1538-7445.AM2017-5259

Abstract 2331: FAM3B/PANDER inhibits cell death and increases tumor growth by modulating the expression of Bcl-2 and Bcl-XL cell survival genes

Abstract FAM3B/PANDER is a novel cytokine-like protein that induces apoptosis in insulin-secreting beta-cells. Since in silico data revealed that FAM3B can be expressed by prostate and breast tumors, we evaluated the putative role of this cytokine in prostate and breast tumor progression. The FAM3B expression was compared by quantitative PCR in LnCAP, PC-3 and DU145 prostate tumor cell lines and MCF-7, ZR-75 and MDA-MB-231 breast tumor cell lines. After treatment with either recombinant FAM3B protein or secreted FAM3B obtained from conditioned media (CM) derived from FAM3B-overexpressing 293T cells , the cell death and viability of DU145 and MDA-MB-231 cell lines were evaluated. DU145 and MDA-MB-231 cells overexpressing FAM3B protein were produced by lentiviral-mediated transduction of full-length FAM3B cDNA. Cell viability and apoptosis were analyzed in DU145-FAM3B and MDA-231-FAM3B cells after treatment with several cell death inducers. Anchorage-independent growth and scratch wound assays were used to evaluate in vitro tumorigenicity and cell migration, respectively. In vivo tumorigenicity and invasiveness were evaluated by tumor xenograft growth in nude mice. We observed that FAM3B was highly expressed by hormone responsive cells (LnCAP and MCF-7) and low expressed in unresponsive hormone cells (PC-3, DU145 and MDA-MB-231). Cell viability and survival of DU145 and MDA-MB-231 cells increased after exogenous treatment with recombinant FAM3B protein or CM containing FAM3B-secreted protein. Overexpression of FAM3B in DU145 and MDA-MB-231 cells promoted an inhibition of apoptosis triggered by TNF-alpha, staurosporine and serum-deprived conditions. Cell death inhibition was accompanied by increased gene expression of the anti-apoptotic Bcl-2 and Bcl-xL genes, and by slight decrease in expression of pro-apoptotic gene Bax and diminished caspases-3, -8 and -9 proteolytic activities. When compared to control, cells overexpressing FAM3B displayed a decreased anchorage independent growth and increased motility in vitro, as well as an increased tumor growth in xenografted nude mice. The immunohistochemistry analysis from tumor xenografts revealed similar anti-apoptotic phenotype displayed by FAM3B-overexpressing tumor cells. Taken together, by activating pro-survival mechanisms, FAM3B overexpression contribute to increased cell death resistance and tumor growth in nude mice, highlighting a putative role for this cytokine in prostate and breast cancer progression. Note: This abstract was not presented at the meeting. Citation Format: Izabela Caldeira, Paula Maciel-Silva, Flavia Ramos Siqueira, Anna Carla Goldberg, Viviane Abreu Nunes, Jose Ernesto Belizario, Humberto Miguel Garay-Malpartida. FAM3B/PANDER inhibits cell death and increases tumor growth by modulating the expression of Bcl-2 and Bcl-XL cell survival genes [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 2331. doi:10.1158/1538-7445.AM2017-2331

Abstract 235: AXL inhibition leads to a reversal of a mesenchymal phenotype sensitizing cancer cells to targeted agents and immuno-oncology therapies

Abstract Mesenchymal properties and the epithelial-to-mesenchymal transition (EMT) contribute to the initiation and progression of many tumor types and ultimately can lead to drug resistance and highly aggressive disease. It is becoming increasingly clear that the more mesenchymal characteristics cancer cells acquire the more resistant they become to standard chemotherapy, targeted agents, and even immune checkpoint inhibitors. We have been exploring the role of the receptor tyrosine kinase, AXL, and its related TAM family members, in promoting the mesenchymal phenotype in cancer cells and how these effects promote drug resistance and escape from immune surveillance. TP-0903, a potent AXL inhibitor, leads to a reversal of the mesenchymal phenotype in multiple cancer models. Following TP-0903 treatment, we observed changes in mRNA expression using RT-qPCR and protein expression using standard immunoblotting that are consistent with a reversal of the mesenchymal phenotype. Upon treatment with TP-0903 cancer cells possessed lower motility and a decrease in anchorage-independent growth, both hallmarks of a mesenchymal cell. In vivo models of erlotinib-resistant non-small cell lung cancer (NSCLC) were utilized to demonstrate TP-0903 single agent activity in highly mesenchymal models; however, more importantly, treatment with TP-0903 was able to sensitize this highly refractory model to erlotinib. AXL function and tumor mesenchymal characteristics also provide mechanisms for the cancer cells to evade immune surveillance. This is achieved by the role that AXL plays in detecting neighboring apoptotic cells resulting in the engulfment of dead cells (efferocytosis) and the associated debris in order to prevent the immune system's exposure to auto-antigens under normal physiological conditions or exposure to cancer-associated neo-antigens in a tumor. Inhibition of AXL by TP-0903 can potentially inhibit tumor-associated efferocytosis leading to a stronger immunogenic response to the tumor. Indeed, results demonstrated synergy when TP-0903 was combined with an anti-PD-L1 agent in a syngeneic triple negative breast cancer mouse model. Interestingly, during the evaluation of TP-0903 in models of EMT, we detected dramatic change in the expression of the retinoic acid (RA) metabolizing protein CYP26A1, suggesting that AXL inhibition leads to changes in RA metabolism. Our data suggest that AXL induces a transition to a mesenchymal phenotype in cancer cells through the suppression of RA signaling and that TP-0903 can rapidly reverse this phenotype by signaling through RA causing the cell to revert to a more differentiated state. Due to its ability to reverse the aggressive mesenchymal phenotype of cancer cells, TP-0903 is a promising agent with the potential to have single agent activity and combined synergy with targeted anti-cancer agents and immunotherapies. Citation Format: Katherine K. Soh, Wontak Kim, Ye Sol Lee, Peter Peterson, Adam Siddiqui-Jain, Steven L. Warner, David J. Bearss, Clifford J. Whatcott. AXL inhibition leads to a reversal of a mesenchymal phenotype sensitizing cancer cells to targeted agents and immuno-oncology therapies. [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 235.

Abstract LB-015: XIAP induction by the MAPK-eIF4G1 pathway drives NFκB activation in inflammatory breast cancer growth and therapeutic resistance

Abstract Inflammatory breast cancer (IBC) is the most lethal, distinct form of breast cancer, however, the basis for its aggressiveness and rapid acquisition of drug resistance is not fully understood. Using immunohistochemical analysis, we identified a strong correlation between high grade, high stage, and triple-negative status and elevated expression of the X-linked inhibitor of apoptosis protein (XIAP) in IBC. Molecular profiling of multiple IBC cell lines revealed that modulating XIAP expression can significantly alter the expression pattern of a 79-gene, characteristic IBC profile that was previously obtained from clinical samples. Using specific antagonists and RNAi, we determined that the mitogen activated protein kinase (MAPK) pathway and its interaction with the protein synthesis initiation factor eIF4G1, both of which are elevated in IBC, cooperate to drive XIAP induction and resistance to therapeutic apoptosis. Further, we found that XIAP expression directly correlates with activation of the transcription factor NFκB, a molecular component defining IBC. Mutational analysis revealed that the BIR1 domain of XIAP is essential for subsequent TAB1:IKKβ-dependent NFκB activation. After determining this association between XIAP and TAB1, we tested a peptide-mediated strategy used to block the BIR1:TAB1 interaction antagonized NFκB activity which led to decreased anchorage independent growth and reversed resistance to an EGFR tyrosine kinase inhibitor. Most significantly, orthotopic implantation of XIAP silenced IBC cells revealed the necessity of expression for IBC tumor growth, while overexpression of XIAP enhanced tumor growth. Our findings establish that XIAP augments the malignant properties in IBC by enhancing NFκB function, identify a druggable pathway with multiple targets, and provide feasibility for the development of novel therapeutics targeting the BIR1 domain of XIAP in IBC. Grant Support: Supported by American Cancer Society, the Duke Cancer Institute as part of the P30 Cancer Center Support Grant NIH CA014236, the Duke Department of Surgery, and DOD grant W81XWH-13-1-0047. Citation Format: Myron K. Evans, Joseph Geradts, Courtney Edwards, Arianna Price, Arjun J. Arora, Amy J. Aldrich, Adrian Ramirez, Timothy J. Robinson, Peter B. Vermeulen, Steven van Laere, Gayathri R. Devi. XIAP induction by the MAPK-eIF4G1 pathway drives NFκB activation in inflammatory breast cancer growth and therapeutic resistance. [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 LB-015.

SSX2 regulates focal adhesion but does not drive the epithelial to mesenchymal transition in prostate cancer

Prostate cancer is the most commonly diagnosed malignancy for men in the United States. Metastatic prostate cancer, the lethal form of the disease, has a life expectancy of approximately five years. Identification of factors associated with this transition to metastatic disease is crucial for future therapies. One such factor is the SSX gene family, a family of cancer/testis antigens (CTA) transcription factors which have been shown to be aberrantly expressed in other cancers and associated with the epithelial to mesenchymal transition (EMT). We have previously shown that SSX expression in prostate cancers was restricted to metastatic tissue and not primary tumors. In this study, we have identified SSX2 as the predominant SSX family member expressed in prostate cancer, and found its expression in the peripheral blood of 19 of 54 (35%) prostate cancer patients, with expression restricted to circulating tumor cells, and in 7 of 15 (47%) metastatic cDNA samples. Further, we examined SSX2 function in prostate cancer through knockdown and overexpression in prostate cancer cell lines. While overexpression had little effect on morphology or gene transcript changes, knockdown of SSX2 resulted in an epithelial morphology, increased cell proliferation, increased expression of genes involved in focal adhesion, decreased anchorage independent growth, increased invasion, and increased tumorigenicity in vivo. We conclude from these findings that SSX2 expression in prostate cancer is not a driver of EMT, but is involved in processes associated with EMT including loss of focal adhesion that may be related to tumor cell dissemination.

Human papillomavirus 16 (HPV16) enhances tumor growth and cancer stemness of HPV-negative oral/oropharyngeal squamous cell carcinoma cells via miR-181 regulation

High-risk human papillomaviruses (e. g., HPV16, HPV18) are closely associated with the development of head and neck cancers including oral/oropharyngeal squamous cell carcinoma (OSCC). We previously demonstrated immortalization of normal human oral keratinocytes by introducing high-risk HPV whole genome, suggesting that HPV infection plays an important role in the early stage of oral carcinogenesis. Although HPV infection may occur in different stages of cancer development, roles of HPV in exacerbating malignant phenotypes in already-transformed cells in the context of cancer stemness are not clearly defined. In this study, we investigated the role of HPV16 in promoting the virulence of HPV-negative OSCC. Introducing HPV16 whole genome in HPV-negative OSCC increased malignant growth and self-renewal capacity, a key characteristic of cancer stem cells (CSCs). HPV16 also enhanced other CSC properties, including aldehyde dehydrogenase 1 (ALDH1) activity, migration/invasion, and CSC-related factor expression. Mechanistically, we found that HPV16 inhibited the expression of miR-181a and miR-181d (miR-181a/d) at the transcriptional level. Ectopic expression of miR-181a/d decreased anchorage independent growth and CSC phenotype of HPV16-transfected OSCC. Furthermore, silencing of miR-181a/d target genes, i. e., K-ras and ALDH1, abrogated the effects of HPV16 in HPV16-transfected OSCC, supporting the functional importance of HPV16/miR-181a/d axis in HPV-mediated oral carcinogenesis. Our study suggests that high-risk HPV infection further promotes malignancy in HPV-negative OSCC by enhancing cancer stemness via miR-181a/d regulation. Consequently, miR-181a/d may represent a novel therapeutic agent for the treatment of HPV-positive OSCC.

Anti-tumour effects of antibodies targeting the extracellular cysteine-rich region of the receptor tyrosine kinase EphB4.

EphB4 is a membrane-bound receptor tyrosine kinase (RTK) commonly over-produced by many epithelial cancers but with low to no expression in most normal adult tissues. EphB4 over-production promotes ligand-independent signaling pathways that increase cancer cell viability and stimulate migration and invasion. Several studies have shown that normal ligand-dependent signaling is tumour suppressive and therefore novel therapeutics which block the tumour promoting ligand-independent signaling and/or stimulate tumour suppressive ligand-dependent signaling will find application in the treatment of cancer. An EphB4-specific polyclonal antibody, targeting a region of 200 amino acids in the extracellular portion of EphB4, showed potent in vitro anti-cancer effects measured by an increase in apoptosis and a decrease in anchorage independent growth. Peptide exclusion was used to identify the epitope targeted by this antibody within the cysteine-rich region of the EphB4 protein, a sequence defined as a potential ligand interacting interface. Addition of antibody to cancer cells resulted in phosphorylation and subsequent degradation of the EphB4 protein, suggesting a mechanism that is ligand mimetic and tumour suppressive. A monoclonal antibody which specifically targets this identified extracellular epitope of EphB4 significantly reduced breast cancer xenograft growth in vivo confirming that EphB4 is a useful target for ligand-mimicking antibody-based anti-cancer therapies.

Diethyl maleate inhibits MCA+TPA transformed cell growth via modulation of GSH, MAPK, and cancer pathways

Murine or human cancer cells have high glutathione levels. Depletion of the elevated GSH inhibits proliferation of cancer cells. Molecular basis for this observation is little understood. In an attempt to find out the underlying mechanism, we reproduced these effects in transformed C3H10T1/2 and BALB/c 3T3 cells using diethyl maleate and studied cytogenomic changes in the whole mouse genome using spotted 8×60K arrays. Transformed cells revealed an increase in GSH levels. GSH depletion by DEM inhibited the growth of transformed cells. The non-cytotoxic dose of DEM (0.25mM) resulted in GSH depletion, ROS generation, cell cycle arrest, apoptosis, decrease in anchorage independent growth, gene expression changes and activation of all three members of the MAPK family. Increase in intracellular GSH levels by GSHe countered the effect of DEM. These results support the physiological importance of GSH in regulation of gene expression for transformed cell growth restraint. This study is of interest in not only understanding the molecular biology of the transformed cells, but also in identifying new targets for development of gene therapy together with the chemotherapy.

KPNA7, a nuclear transport receptor, promotes malignant properties of pancreatic cancer cells in vitro

Pancreatic cancer is an aggressive malignancy and one of the leading causes of cancer deaths. The high mortality rate is mostly due to the lack of appropriate tools for early detection of the disease and a shortage of effective therapies. We have previously shown that karyopherin alpha 7 (KPNA7), the newest member of the alpha karyopherin family of nuclear import receptors, is frequently amplified and overexpressed in pancreatic cancer. Here, we report that KPNA7 expression is absent in practically all normal human adult tissues but elevated in several pancreatic cancer cell lines. Inhibition of KPNA7 expression in AsPC-1 and Hs700T pancreatic cancer cells led to a reduction in cell growth and decreased anchorage independent growth, as well as increased autophagy. The cell growth effects were accompanied by an induction of the cell cycle regulator p21 and a G1 arrest of the cell cycle. Interestingly, the p21 induction was caused by increased mRNA synthesis and not defective nuclear transport. These data strongly demonstrate that KPNA7 silencing inhibits the malignant properties of pancreatic cancer cells in vitro and thereby provide the first evidence on the functional role for KPNA7 in human cancer.

Gli1 Transcriptional Activity is Negatively Regulated by AKT2 in Neuroblastoma

Activation of the Hedgehog (Hh) signaling pathway has been implicated in a variety of malignancies including neuroblastoma. Expression of Gli1, a downstream effector of Hh, correlates with a favorable prognosis in patients with neuroblastoma. Moreover, Gli1 overexpression reduces mitotic index and induces transcription of genes involved in the differentiation of neuroblastoma cells; however, much remains unknown regarding the regulation of Gli1 transcriptional activity. Here, we report a novel negative regulation of Gli1 transcriptional activity by PI3K/AKT2 signal transduction pathway. Constitutively active PI3K subunit, p110α, inhibited Gli1 transcriptional activity in neuroblastoma cells, whereas, overexpression of an inactive form of PI3K subunit, p85, enhanced its activity. Specifically, the AKT2 isoform inhibited Gli1 luciferase activity. Silencing AKT2 using siRNA increased Gli1 transcriptional activity and conversely, overexpression of constitutively active AKT2 (myr-AKT2) decreased Gli1 transcriptional activity. Furthermore, Gli1 overexpression-mediated decrease in anchorage-independent growth was rescued by AKT2 overexpression. We also demonstrated that AKT2 overexpression regulates the nuclear-cytoplasmic distribution of exogenous Gli1 protein in neuroblastoma cells by relieving a GSK3β-mediated destabilization of SUFU, a negative regulator of Gli1 nuclear translocation. Inhibition of nuclear Gli1 accumulation may explain for the suppression of the tumor-suppressive function of Gli1. Collectively, our findings suggest an important role of Gli1 as a tumor suppressor in neuroblastoma, and offer a mechanism by which AKT2 regulates the subcellular localization, and in turn, inhibits the tumor-suppressive function of Gli1 in neuroblastoma.

Abstract 1089: Spontaneous formation of sarcomas in Usp18 knockout mice.

Abstract Undifferentiated pleomorphic sarcoma (UPS) and leimyosarcoma are two of the most common adult soft tissue sarcomas. Their etiology is unknown hence the need for new models to elucidate the biology of these diseases. We report here a spontaneously occurring mouse model of sarcoma in Usp18 knockout mice. Usp18 is the deconjugase for isgylation. Isgylation is the ubiquitin-like protein modification in which ISG15 is conjugated to proteins. Usp18 removes ISG15 from the target proteins, thus reversing the functional consequences of ISG15 conjugation. Usp18 knockout mice on a C57Bl6 background developed neurological abnormalities and exhibited 100% mortality within 5 months. These mice were also hypersensitive to Type I IFN stimulation, which was attributed to an increased JAK-STAT signaling. We now report that Usp18 knockout mice in a FVB background developed subcutaneous tumors in the neck and trunk regions from ages ranging from 6 months to 16 months. Tumor formation to date shows a penetrance of 77%. Immunohistochemical analysis characterized the tumors as either UPSs or leimyosarcomas. About 30% of these tumors displayed stabilization of p53, a common event also seen in human sarcomas. We derived two cell lines (KHC-1, KHC-2) from the sarcomas of separate mice, and analyzed the levels of STAT family proteins in these cell lines. Stat3 was noted to be constitutively activated in these sarcoma cell lines. Persistent activation of Stat3 is seen in diverse human malignancies such as breast and head and neck cancers. To determine if restoring Usp18 activity reverses tumorigenicity in these cells, human USP18 was introduced retrovirally. Cells re-expressing USP18 versus empty vector showed a substantial decrease in anchorage independent growth in soft agar and also had reduced levels of activated Stat3 (pStat3). Furthermore, treatment of the parental cell lines with a Stat3 inhibitor, JSI-124, led to decreased cell growth, apoptosis and reduced levels of pStat3. In summary, Usp18 knockout mice spontaneously develop sarcomas, whose tumorigenicity can be partially reversed by reintroduction of Usp18. Thus, this new and tractable model should prove useful to elucidate the activity of Stat3 targeting and other agents to treat sarcomas. Citation Format: Fadzai Chinyengetere, Andrew Giustini, David Sekula, Sarah Freemantle, Vincent A. Memoli, Paul Jack Hoopes, Ethan Dmitrovsky. Spontaneous formation of sarcomas in Usp18 knockout mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1089. doi:10.1158/1538-7445.AM2013-1089

Abstract 3715: Zoledronic acid inhibits cancer growth and cancer stem cell phenotypes in head and neck squamous cell carcinoma.

Abstract Zoledronic acid (ZA), a third generation of bisphosphonates, is known to have both anti-osteolytic and anti-tumor activities. It exerts anti-osteolytic function by interfering with mevalonate pathway and preventing protein prenylation required for growth and survival in osteoclast cells. Also, similar function has been found to induce cancer apoptosis and inhibit cancer growth in bone-related cancer, but its effect on other cancer types, including head and neck squamous cell carcinoma (HNSCC) is still unclear. In this study, we first monitored the effect of ZA on cancer growth in HNSCC cell lines in vitro. ZA significantly suppressed cancer growth, shown by decreased anchorage independent growth in soft agar assay and diminished cell growth in organotypic raft culture. Because cancer stem cell (CSC; also called tumor-initiating cell) plays a significant role in cancer growth, we further investigated the effect of ZA on CSC in HNSCC. ZA-treated HNSCC cells significantly decreased CSC phenotypes such as sphere forming ability and migratory ability. In SCC4, a tongue squamous cell carcinoma cell line, side population and expression of pluripotency marker genes were significantly decreased by ZA. Also, ZA inhibited the CSC-related Sonic Hedgehog pathway in SCC4 as demonstrated by decreased mRNA expression of transcriptional factor, Gli-1. Taken together, our data suggest that ZA targets CSC and hence inhibits cancer growth in HNSCC. Citation Format: Sung Hee Lee, Reuben Kim, Mo Kang, No-Hee Park, Ki-Hyuk Shin. Zoledronic acid inhibits cancer growth and cancer stem cell phenotypes in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3715. doi:10.1158/1538-7445.AM2013-3715

Abstract 1753: KPNA7, a nuclear transport receptor, promotes malignant properties of pancreatic cancer cells in vitro.

Abstract Pancreatic cancer is an aggressive malignancy and one of the leading causes of cancer deaths. The high mortality rate is mostly due to the lack of appropriate tools for the early detection of the disease and a shortage of effective therapies. We have previously shown that karyopherin alpha 7 (KPNA7) is frequently amplified and overexpressed in pancreatic cancer. KPNA7 is a recently characterized member of the alpha karyopherin family of nuclear import receptors. The nuclear import machinery is composed of a complex network of proteins required for the appropriate transport of proteins from the cytoplasm to the nucleus. Abnormal function of this machinery leads to incorrect localization of nuclear proteins and disturbances in cellular homeostasis, which may subsequently contribute to the development of various diseases including cancer. Indeed, previous evidence has linked members of the karyopherin alpha family, especially KPNA2, to cancer pathogenesis. In this study we demonstrate that KPNA7 expression is absent in practically all normal human adult tissues but elevated in several pancreatic cancer cell lines as well as in some other cancer types. Inhibition of KPNA7 expression in AsPC-1 and Hs700T pancreatic cancer cells led to a striking reduction in cell growth due to the induction of the cell cycle regulator p21 and subsequent G1 arrest of the cell cycle. In both of these cell lines KPNA7 silencing also resulted in decreased anchorage independent growth, and additionally in the AsPC-1 cells reduced cell migration and invasion. Furthermore, in the Hs700T cells KPNA7 silencing resulted in dramatic morphological change where the cells acquired fibroblast-like shape. This phenotypic change was not explained by induction of EMT or senescence. In conclusion, our data strongly demonstrate that KPNA7 silencing inhibits the malignant properties of pancreatic cancer cells in vitro and thereby provide the first evidence on the functional role of KPNA7 in human cancer. Moreover, the lack of KPNA7 expression in normal adult tissues highlights it as a potential novel therapeutic target for cancer. Further studies are needed to identify the cargo proteins transported by KPNA7. These studies are likely to reveal important new information on nuclear transport and may highlight the molecular mechanisms involved in KPNA7 mediated phenotypes in cancer cells. Citation Format: Hanna E. Rauhala, Eeva Laurila, Elisa Vuorinen, Kimmo Savinainen, Anne Kallioniemi. KPNA7, a nuclear transport receptor, promotes malignant properties of pancreatic cancer cells in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1753. doi:10.1158/1538-7445.AM2013-1753