Role of stromal activin A in human pancreatic cancer and metastasis in mice.

Pancreatic ductal adenocarcinoma (PDAC) has extensive stromal involvement and remains one of the cancers with the highest mortality rates. Activin A has been implicated in colon cancer and its stroma but its role in the stroma of PDAC has not been elucidated. Activin A expression in cancer and stroma was assessed in human PDAC tissue microarrays (TMA). Activin A expression in human TMA is significantly higher in cancer samples, with expression in stroma correlated with shorter survival. Cultured pancreatic stellate cells (PSC) were found to secrete high levels of activin A resulting in PDAC cell migration that is abolished by anti-activin A neutralizing antibody. KPC mice treated with anti-activin A neutralizing antibody were evaluated for tumors, lesions and metastases quantified by immunohistochemistry. KPC mice with increased tumor burden express high plasma activin A. Treating KPC mice with an activin A neutralizing antibody does not reduce primary tumor size but decreases tumor metastases. From these data we conclude that PDAC patients with high activin A expression in stroma have a worse prognosis. PSCs secrete activin A, promoting increased PDAC migration. Inhibition of activin A in mice decreased metastases. Hence, stroma-rich PDAC patients might benefit from activin A inhibition.

Author Correction: Increased stiffness of the tumor microenvironment in colon cancer stimulates cancer associated fibroblast-mediated prometastatic activin A signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Activin A Modulates Inflammation in Acute Pancreatitis and Strongly Predicts Severe Disease Independent of Body Mass Index.

INTRODUCTION: Acute pancreatitis (AP) is a healthcare challenge with considerable mortality. Treatment is limited to supportive care, highlighting the need to investigate disease drivers and prognostic markers. Activin A is an established mediator of inflammatory responses, and its serum levels correlate with AP severity. We hypothesized that activin A is independent of body mass index (BMI) and is a targetable promoter of the AP inflammatory response. METHODS: We assessed whether BMI and serum activin A levels are independent markers to determine disease severity in a cohort of patients with AP. To evaluate activin A inhibition as a therapeutic, we used a cerulein-induced murine model of AP and treated mice with activin A-specific neutralizing antibody or immunoglobulin G control, both before and during the development of AP. We measured the production and release of activin A by pancreas and macrophage cell lines and observed the activation of macrophages after activin A treatment. RESULTS: BMI and activin A independently predicted severe AP in patients. Inhibiting activin A in AP mice reduced disease severity and local immune cell infiltration. Inflammatory stimulation led to activin A production and release by pancreas cells but not by macrophages. Macrophages were activated by activin A, suggesting activin A might promote inflammation in the pancreas in response to injury. DISCUSSION: Activin A provides a promising therapeutic target to interrupt the cycle of inflammation and tissue damage in AP progression. Moreover, assessing activin A and BMI in patients on hospital admission could provide important predictive measures for screening patients likely to develop severe disease.

Nuclear translocation of Atox1 potentiates activin A-induced cell migration and colony formation in colon cancer.

BACKGROUND: Colorectal cancer remains a deadly cancer due to metastatic disease. To understand the molecular mechanisms of metastasis in colon cancer, we investigated whether the copper chaperone antioxidant-1 (Atox1) protein plays a role in this process. Recent findings indicate that Atox1 protein has transcription factor activities and plays a vital role in cell proliferation in cancer cells. However, the role of Atox1 in metastasis has not been examined. METHODS: Atox1 expression was determined by immunofluorescence in a tissue microarray generated from a spectrum of CRC patients. Subcellular fractionation of colon cancer cell lines SW480 and SW620 cells was used to examine the cellular location of Atox1 in the face of activin A, a cytokine that stimulates colon cancer metastasis. Atox1 expression was genetically manipulated and cellular migration measured through trans-well assay and proliferation measured by colony formation assays. RESULTS: Here we demonstrate that in patients with metastatic colon cancer, there is a significant increase in the expression of nuclear Atox1. Interestingly, the metastatic CRC cell line SW620 has increased nuclear localization of Atox1 compared to its related non-metastatic cell line SW480. Further, inhibition of endogenous Atox1 by siRNA in SW620 decreased colony formation and reactive oxygen species generation via decreased expression of Atox1 targets cyclin D1 and NADPH oxidase subunit p47 phox, respectively. Additionally, overexpression of nuclear-targeted but not copper binding domain-mutated Atox1 in SW480 cells increased colony formation and cell migration that was further augmented by activin A stimulation, a known enhancer of colon cancer metastasis. CONCLUSIONS: Our findings suggest that nuclear Atox1 might be a new therapeutic target as well as a new biomarker for metastatic colorectal cancer.

Increased stiffness of the tumor microenvironment in colon cancer stimulates cancer associated fibroblast-mediated prometastatic activin A signaling.

Colorectal cancer (CRC) is the second deadliest cancer in the US due to its propensity to metastasize. Stromal cells and especially cancer-associated fibroblasts (CAF) play a critical biophysical role in cancer progression, but the precise pro-metastatic mechanisms are not clear. Activin A, a TGF-ß family member, is a strong pro-metastatic cytokine in the context of CRC. Here, we assessed the link between biophysical forces and pro-metastatic signaling by testing the hypothesis that CAF-generated mechanical forces lead to activin A release and associated downstream effects. Consistent with our hypothesis, we first determined that stromal activin A secretion increased with increasing substrate stiffness. Then we found that stromally-secreted activin A induced ligand-dependent CRC epithelial cell migration and epithelial to mesenchymal transition (EMT). In addition, serum activin A levels are significantly increased in metastatic (stage IV) CRC patients (1.558 ng/ml versus 0.4179 ng/ml, p < 0.05). We propose that increased tumor microenvironment stiffness leads to stromal cell-mediated TGF-ß family signaling relying on the induction and utilization of activin A signaling.

TGFß and activin A in the tumor microenvironment in colorectal cancer.

Although overall survival in colorectal cancer (CRC) is increasing steadily due to progress in screening, therapeutic options and precise diagnostic tools remain scarce. As the understanding of CRC as a complex and multifactorial condition moves forward, the tumor microenvironment has come into focus as a source of diagnostic markers and potential therapeutic targets. The role of TGFß in shifting the epithelial cancer compartment towards invasiveness and a pro-migratory phenotype via stromal signaling has been widely investigated. Accordingly, recent studies have proposed that CRC patients could be stratified into distinct subtypes and have identified one poor prognosis subset of CRC that is characterized by high stromal activity and elevated levels of TGFß. The TGFß superfamily member activin A is crucial for the pro-metastatic properties of the TGFß pathway, yet it has been under-researched in CRC carcinogenesis. In this review, we will elucidate the signaling network and interdependency of both ligands in the context of the tumor microenvironment in CRC.

Activin in acute pancreatitis: Potential risk-stratifying marker and novel therapeutic target.

Acute Pancreatitis is a substantial health care challenge with increasing incidence. Patients who develop severe disease have considerable mortality. Currently, no reliable predictive marker to identify patients at risk for severe disease exists. Treatment is limited to rehydration and supporting care suggesting an urgent need to develop novel approaches to improve standard care. Activin is a critical modulator of inflammatory responses, but has not been assessed in pancreatitis. Here, we demonstrate that serum activin is elevated and strongly correlates with disease severity in two established murine models of acute pancreatitis induced by either cerulein or IL-12 + IL-18. Furthermore, in mice, inhibition of activin conveys survival benefits in pancreatitis. In addition, serum activin levels were measured from a retrospective clinical cohort of pancreatitis patients and high activin levels in patients at admission are predictive of worse outcomes, indicated by longer overall hospital and intensive care unit stays. Taken together, activin is a novel candidate as a clinical marker to identify those acute pancreatitis patients with severe disease who would benefit from aggressive treatment and activin may be a therapeutic target in severe acute pancreatitis.

Increased Frequency of KRAS Mutations in African Americans Compared with Caucasians in Sporadic Colorectal Cancer.

OBJECTIVES: The basis for over-representation of colorectal cancer (CRC) in African-American (AA) populations compared with Caucasians are multifactorial and complex. Understanding the mechanisms for this racial disparity is critical for delivery of better care. Several studies have investigated sporadic CRC for differences in somatic mutations between AAs and Caucasians, but owing to small study sizes and conflicting results to date, no definitive conclusions have been reached. METHODS: Here, we present the first systematic literature review and meta-analysis investigating the mutational differences in sporadic CRC between AAs and Caucasians focused on frequent driver mutations (APC,TP53, KRAS,PI3CA, FBXW7,SMAD4, and BRAF). Publication inclusion criteria comprised sporadic CRC, human subjects, English language, information on ethnicity (AA, Caucasian, or both), total subject number >20, and information on mutation frequencies. RESULTS: We identified 6,234 publications. Meta-analysis for APC, TP54, FBXW7, or SMAD4 was not possible owing to paucity of data. KRAS mutations were statistically less frequent in non-Hispanic Whites when compared with AAs (odds ratio, 0.640; 95% confidence interval (CI): 0.5342-0.7666; P=0.0001), while the mutational differences observed in BRAF and PI3CA did not reach statistical significance. CONCLUSIONS: Here, we report the mutational patterns for KRAS, BRAF, and PI3CA in sporadic CRC of AAs and Caucasians in a systematic meta-analysis of previously published data. We identified an increase in KRAS mutations in sporadic CRC in AAs, which may contribute to worse prognosis and increased mortality of CRC in AAs. Future studies investigating health-care disparities in CRC in AAs should control for KRAS mutational frequency.

Activin signaling is an essential component of the TGF-ß induced pro-metastatic phenotype in colorectal cancer.

Advanced colorectal cancer (CRC) remains a critical health care challenge worldwide. Various TGF-ß superfamily members are important in colorectal cancer metastasis, but their signaling effects and predictive value have only been assessed in isolation. Here, we examine cross-regulation and combined functions of the two most prominent TGF-ß superfamily members activin and TGF-ß in advanced colorectal cancer. In two clinical cohorts we observed by immune-based assay that combined serum and tissue activin and TGF-ß ligand levels predicts outcome in CRC patients and is superior to single ligand assessment. While TGF-ß growth suppression is independent of activin, TGF-ß treatment leads to increased activin secretion in colon cancer cells and TGF-ß induced cellular migration is dependent on activin, indicating pathway cross-regulation and functional interaction in vitro. mRNA expression of activin and TGF-ß pathway members were queried in silico using the TCGA data set. Coordinated ligand and receptor expression is common in solid tumors for activin and TGF-ß pathway members. In conclusion, activin and TGF-ß are strongly connected signaling pathways that are important in advanced CRC. Assessing activin and TGF-ß signaling as a unit yields important insights applicable to future diagnostic and therapeutic interventions.

NFkB is essential for activin-induced colorectal cancer migration via upregulation of PI3K-MDM2 pathway.

Colorectal cancer (CRC) remains a common and deadly cancer due to metastatic disease. Activin and TGFB (TGFß) signaling are growth suppressive pathways that exert non-canonical pro-metastatic effects late in CRC carcinogenesis. We have recently shown that activin downregulates p21 via ubiquitination and degradation associated with enhanced cellular migration independent of SMADs. To investigate the mechanism of metastatic activin signaling, we examined activated NFkB signaling and activin ligand expression in CRC patient samples and found a strong correlation. We hypothesize that activation of the E3 ubiquitin ligase MDM2 by NFkB leads to p21 degradation in response to activin treatment. To dissect the link between activin and pro-carcinogenic NFkB signaling and downstream targets, we found that activin but not TGFB induced activation of NFkB leading to increased MDM2 ubiquitin ligase via PI3K. Further, overexpression of wild type p65 NFkB increased MDM2 expression while the NFkB inhibitors NEMO-binding domain (NBD) and Bay11-7082 blocked the activin-induced increase in MDM2. In conclusion, in colon cancer cell migration, activin utilizes NFkB to induce MDM2 activity leading to the degradation of p21 in a PI3K dependent mechanism. This provides new mechanistic knowledge linking activin and NFkB signaling in advanced colon cancer which is applicable to targeted therapeutic interventions.

Expression of an Oncogenic BARD1 Splice Variant Impairs Homologous Recombination and Predicts Response to PARP-1 Inhibitor Therapy in Colon Cancer.

BRCA1-associated RING domain protein 1 (BARD1) stabilizes BRCA1 protein by forming a heterodimeric RING-RING complex, and impacts function of BRCA1, including homologous recombination (HR) repair. Although colon cancer cells usually express wild type BRCA1, presence of an oncogenic BARD1 splice variant (SV) in select cancers may render BRCA1 dysfunctional and allow cells to become sensitive to HR targeting therapies. We previously reported association of loss of full-length (FL) BARD1 with poor prognosis in colon cancer as well as expression of various BARD1 SVs with unknown function. Here we show that loss of BARD1 function through the expression of a BARD1 SV, BARD1ß, results in a more malignant phenotype with decreased RAD51 foci formation, reduced BRCA1 E3 ubiquitin ligase activity, and decreased nuclear BRCA1 protein localization. BARD1ß sensitizes colon cancer cells to poly ADP ribose polymerase 1 (PARP-1) inhibition even in a FL BRCA1 background. These results suggest that expression of BARD1ß may serve as a future biomarker to assess suitability of colon cancers for HR targeting with PARP-1 inhibitors in treatment of advanced colon cancer.

Identification of potential glucocorticoid receptor therapeutic targets in multiple myeloma.

Glucocorticoids (GC) are a cornerstone of combination therapies for multiple myeloma. However, patients ultimately develop resistance to GCs frequently based on decreased glucocorticoid receptor (GR) expression. An understanding of the direct targets of GC actions, which induce cell death, is expected to culminate in potential therapeutic strategies for inducing cell death by regulating downstream targets in the absence of a functional GR. The specific goal of our research is to identify primary GR targets that contribute to GC-induced cell death, with the ultimate goal of developing novel therapeutics around these targets that can be used to overcome resistance to GCs in the absence of GR. Using the MM.1S glucocorticoid-sensitive human myeloma cell line, we began with the broad platform of gene expression profiling to identify glucocorticoid-regulated genes further refined by combination treatment with phosphatidylinositol-3'-kinase inhibition (PI3Ki). To further refine the search to distinguish direct and indirect targets of GR that respond to the combination GC and PI3Ki treatment of MM.1S cells, we integrated 1) gene expression profiles of combination GC treatment with PI3Ki, which induces synergistic cell death; 2) negative correlation between genes inhibited by combination treatment in MM.1S cells and genes over-expressed in myeloma patients to establish clinical relevance and 3) GR chromatin immunoprecipitation with massively parallel sequencing (ChIP-Seq) in myeloma cells to identify global chromatin binding for the glucocorticoid receptor (GR). Using established bioinformatics platforms, we have integrated these data sets to identify a subset of candidate genes that may form the basis for a comprehensive picture of glucocorticoid actions in multiple myeloma. As a proof of principle, we have verified two targets, namely RRM2 and BCL2L1, as primary functional targets of GR involved in GC-induced cell death.

Identification of p38ß as a therapeutic target for the treatment of Sézary syndrome.

Cutaneous T-cell lymphomas (CTCLs) represent a group of hematopoietic malignancies that home to the skin and have no known molecular basis for disease pathogenesis. Sézary syndrome (SS) is the leukemic variant of CTCL. Currently, CTCL is incurable, highlighting the need for new therapeutic modalities. We have previously observed that combined small-molecule inhibition of protein kinase C-ß (PKCß) and glycogen synthase kinase 3 (GSK3) causes synergistic apoptosis in CTCL cell lines and patient cells. Through microarray analysis of a SS cell line, we surveyed global gene expression following combined PKCß-GSK3 treatment to elucidate therapeutic targets responsible for cell death. Clinically relevant targets were defined as genes differentially expressed in SS patients that were modulated by combination-drug treatment of SS cells. Gene set enrichment analysis uncovered candidate genes enriched for an immune-cell signature, specifically the T-cell receptor and mitogen-activated protein kinase signaling pathways. Further analysis identified p38 as a potential therapeutic target that is overexpressed in SS patients and decreased by synergistic-inhibitor treatment. This target was verified through small-molecule inhibition of p38, leading to cell death in both SS cell lines and patient cells. These data establish p38 as a SS biomarker and a potential therapeutic target for the treatment of CTCL.

Clinical perspective on chemo-resistance and the role of RNA processing.

Pre-messenger RNA splicing is significantly changed in cancer cells leading to the expression of cancer-specific transcripts. These transcripts have the potential to be used as cancer biomarkers and also as targets for new therapeutic approaches. In addition, the cancer-specific transcripts have the potential to alter the drug response of the cancer cells creating a chemo-resistant state. This later property of alternative splicing presents a challenge to clinicians in the design of effective therapeutic regimens. When a patient's cancer relapses it is frequently refractory to standard chemotherapies resulting in a poor clinical outcome. Therefore, understanding the mechanisms of how alternative splicing can lead to chemo-resistance is critical to the effective delivery of treatment. Here, we will discuss the impact of alternative splicing variants on drug metabolism and activation; on drug interactions with cell signaling pathways; and on cell death pathways in cancer therapeutics. In addition to the initial characterization of splicing variants, the mechanisms leading to alterations in splicing are being studied in the setting of chemo-resistance and will be discussed here. The promise of therapeutic intervention to obviate the impact of these splicing variants will significantly enhance treatment options for cancer patients.

An epithelial-mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance.

PURPOSE: Epithelial-mesenchymal transition (EMT) has been associated with metastatic spread and EGF receptor (EGFR) inhibitor resistance. We developed and validated a robust 76-gene EMT signature using gene expression profiles from four platforms using non-small cell lung carcinoma (NSCLC) cell lines and patients treated in the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) study. EXPERIMENTAL DESIGN: We conducted an integrated gene expression, proteomic, and drug response analysis using cell lines and tumors from patients with NSCLC. A 76-gene EMT signature was developed and validated using gene expression profiles from four microarray platforms of NSCLC cell lines and patients treated in the BATTLE study, and potential therapeutic targets associated with EMT were identified. RESULTS: Compared with epithelial cells, mesenchymal cells showed significantly greater resistance to EGFR and PI3K/Akt pathway inhibitors, independent of EGFR mutation status, but more sensitivity to certain chemotherapies. Mesenchymal cells also expressed increased levels of the receptor tyrosine kinase Axl and showed a trend toward greater sensitivity to the Axl inhibitor SGI-7079, whereas the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing Axl and in a xenograft model of mesenchymal NSCLC. In patients with NSCLC, the EMT signature predicted 8-week disease control in patients receiving erlotinib but not other therapies. CONCLUSION: We have developed a robust EMT signature that predicts resistance to EGFR and PI3K/Akt inhibitors, highlights different patterns of drug responsiveness for epithelial and mesenchymal cells, and identifies Axl as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype.

8-Amino-adenosine activates p53-independent cell death of metastatic breast cancers.

8-Amino-adenosine (8-NH(2)-Ado) is a ribose sugar nucleoside analogue that reduces cellular ATP levels and inhibits mRNA synthesis. Estrogen receptor-negative (ER-) metastatic breast cancers often contain mutant p53; therefore, we asked if 8-NH(2)-Ado could kill breast cancer cells without activating the p53-pathway. Regardless of the breast cancer subtype tested or the p53 status of the cells, 8-NH(2)-Ado was more cytotoxic than either gemcitabine or etoposide. 8-NH(2)-Ado treatment inhibited cell proliferation, activated cell death, and did not activate transcription of the p53 target gene p21 or increase protein levels of either p53 or p21. This occurred in the estrogen receptor-positive (ER+) MCF-7 cells that express wild-type p53, the ER+ T47-D cells that express mutant p53, and the ER- MDA-MB-468 cells or MDA-MB-231 cells that both express mutant p53. 8-NH(2)-Ado induced apoptotic death of MCF-7 cells and apoptosis was not inhibited by knockdown of functional p53. Moreover, the pan-caspase inhibitor Z-VAD blocked the 8-NH(2)-Ado-induced MCF-7 cell death. Interestingly, 8-NH(2)-Ado caused the MDA-MB-231 cells to detach from the plate with only limited evidence of apoptotic cell death markers and the cell death was not inhibited by Z-VAD. Inhibition of MDA-MB-231 cell autophagy, by reduction of ATG7 or 3-methyladenine treatment, did not block this 8-NH(2)-Ado-mediated cytotoxicity. Importantly 8-NH(2)-Ado was highly cytotoxic to triple-negative breast cancer cells and worked through a pathway that did not require wild-type p53 for cytoxicity. Therefore, 8-NH(2)-Ado should be considered for the treatment of triple-negative breast cancers that are chemotherapy resistant.

Integrative gene expression profiling reveals G6PD-mediated resistance to RNA-directed nucleoside analogues in B-cell neoplasms.

The nucleoside analogues 8-amino-adenosine and 8-chloro-adenosine have been investigated in the context of B-lineage lymphoid malignancies by our laboratories due to the selective cytotoxicity they exhibit toward multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL) cell lines and primary cells. Encouraging pharmacokinetic and pharmacodynamic properties of 8-chloro-adenosine being documented in an ongoing Phase I trial in CLL provide additional impetus for the study of these promising drugs. In order to foster a deeper understanding of the commonalities between their mechanisms of action and gain insight into specific patient cohorts positioned to achieve maximal benefit from treatment, we devised a novel two-tiered chemoinformatic screen to identify molecular determinants of responsiveness to these compounds. This screen entailed: 1) the elucidation of gene expression patterns highly associated with the anti-tumor activity of 8-chloro-adenosine in the NCI-60 cell line panel, 2) characterization of altered transcript abundances between paired MM and MCL cell lines exhibiting differential susceptibility to 8-amino-adenosine, and 3) integration of the resulting datasets. This approach generated a signature of seven unique genes including G6PD which encodes the rate-determining enzyme of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase. Bioinformatic analysis of primary cell gene expression data demonstrated that G6PD is frequently overexpressed in MM and CLL, highlighting the potential clinical implications of this finding. Utilizing the paired sensitive and resistant MM and MCL cell lines as a model system, we go on to demonstrate through loss-of-function and gain-of-function studies that elevated G6PD expression is necessary to maintain resistance to 8-amino- and 8-chloro-adenosine but insufficient to induce de novo resistance in sensitive cells. Taken together, these results indicate that G6PD activity antagonizes the cytotoxicity of 8-substituted adenosine analogues and suggests that administration of these agents to patients with B-cell malignancies exhibiting normal levels of G6PD expression may be particularly efficacious.

Multiple myeloma exhibits novel dependence on GLUT4, GLUT8, and GLUT11: implications for glucose transporter-directed therapy.

Multiple myeloma is one of numerous malignancies characterized by increased glucose consumption, a phenomenon with significant prognostic implications in this disease. Few studies have focused on elucidating the molecular underpinnings of glucose transporter (GLUT) activation in cancer, knowledge that could facilitate identification of promising therapeutic targets. To address this issue, we performed gene expression profiling studies involving myeloma cell lines and primary cells as well as normal lymphocytes to uncover deregulated GLUT family members in myeloma. Our data demonstrate that myeloma cells exhibit reliance on constitutively cell surface-localized GLUT4 for basal glucose consumption, maintenance of Mcl-1 expression, growth, and survival. We also establish that the activities of the enigmatic transporters GLUT8 and GLUT11 are required for proliferation and viability in myeloma, albeit because of functionalities probably distinct from whole-cell glucose supply. As proof of principle regarding the therapeutic potential of GLUT-targeted compounds, we include evidence of the antimyeloma effects elicited against both cell lines and primary cells by the FDA-approved HIV protease inhibitor ritonavir, which exerts a selective off-target inhibitory effect on GLUT4. Our work reveals critical roles for novel GLUT family members and highlights a therapeutic strategy entailing selective GLUT inhibition to specifically target aberrant glucose metabolism in cancer.