Judith C. Sporn

Histone macroH2A isoforms predict the risk of lung cancer recurrence

Lung cancer is the leading cause of cancer deaths. Despite optimal diagnosis and early treatment, many patients die of recurrent disease. There are no sufficiently useful biomarkers to predict the risk of tumor recurrence. Here, we show that expression of histone macroH2A1.1 and macroH2A2 predicts lung cancer recurrence, identifying these histone variants as a novel tool for an improved risk stratification of cancer patients. Moreover, macroH2A isoforms are highly expressed in cells undergoing senescence, a known antitumor mechanism, suggesting macroH2A1.1 may be a useful biomarker for senescent cells in tumors.

Differential Regulation and Predictive Potential of MacroH2A1 Isoforms in Colon Cancer

Histone variant macroH2A1 has two splice isoforms, macroH2A1.1 and macroH2A1.2, with tissue- and cell-specific expression patterns. Although macroH2A1.1 is mainly found in differentiated, nonproliferative tissues, macroH2A1.2 is more generally expressed, including in tissues with ongoing cell proliferation. Consistently, studies in breast and lung cancer have demonstrated a strong correlation between macroH2A1.1 levels and proliferation, which is not the case for macroH2A1.2. This is the first study to assess the differential regulation and predictive potential of macroH2A1 isoforms in colon cancer. We found that macroH2A1.1 mRNA was down-regulated in primary colorectal cancer samples compared to matched normal colon tissue, whereas macroH2A1.2 was up-regulated. At the protein level, down-regulation of macroH2A1.1 correlated significantly with patient outcome (P = 0.0012), and loss of macroH2A1.1 was associated with a worse outcome. Over the course of Caco-2 cell differentiation, macroH2A1.1 was up-regulated at both the RNA and protein levels, whereas macroH2A1.2 was slightly down-regulated at the RNA level and stable at the protein level. These changes were accompanied by an antiproliferative phenotype exhibiting features of cellular senescence. Loss of macroH2A1.1 in vitro was characterized by a phenotype associated with cell growth and metastasis. These data demonstrate that macroH2A1 isoforms are differentially regulated in colon cancer, reflecting the degree of cellular differentiation. Notably, macroH2A1.1 expression predicts survival in colon cancer, thus identifying macroH2A1.1 as a novel colon cancer biomarker.

Effects of Activin and TGFβ on p21 in Colon Cancer

Activin and TGFβ share SMAD signaling and colon cancers can inactivate either pathway alone or simultaneously. The differential effects of activin and TGFβ signaling in colon cancer have not been previously dissected. A key downstream target of TGFβ signaling is the cdk2 inhibitor p21 (p21cip1/waf1). Here, we evaluate activin-specific effects on p21 regulation and resulting functions. We find that TGFβ is a more potent inducer of growth suppression, while activin is a more potent inducer of apoptosis. Further, growth suppression and apoptosis by both ligands are dependent on SMAD4. However, activin downregulates p21 protein in a SMAD4-independent fashion in conjunction with increased ubiquitination and proteasomal degradation to enhance migration, while TGFβ upregulates p21 in a SMAD4-dependent fashion to affect growth arrest. Activin-induced growth suppression and cell death are dependent on p21, while activin-induced migration is counteracted by p21. Further, primary colon cancers show differential p21 expression consistent with their ACVR2/TGFBR2 receptor status. In summary, we report p21 as a differentially affected activin/TGFβ target and mediator of ligand-specific functions in colon cancer, which may be exploited for future risk stratification and therapeutic intervention.

Activin Signaling in Microsatellite Stable Colon Cancers Is Disrupted by a Combination of Genetic and Epigenetic Mechanisms

Background Activin receptor 2 (ACVR2) is commonly mutated in microsatellite unstable (MSI) colon cancers, leading to protein loss, signaling disruption, and larger tumors. Here, we examined activin signaling disruption in microsatellite stable (MSS) colon cancers. Methods Fifty-one population-based MSS colon cancers were assessed for ACVR1, ACVR2 and pSMAD2 protein. Consensus mutation-prone portions of ACVR2 were sequenced in primary cancers and all exons in colon cancer cell lines. Loss of heterozygosity (LOH) was evaluated for ACVR2 and ACVR1, and ACVR2 promoter methylation by methylation-specific PCR and bisulfite sequencing and chromosomal instability (CIN) phenotype via fluorescent LOH analysis of 3 duplicate markers. ACVR2 promoter methylation and ACVR2 expression were assessed in colon cancer cell lines via qPCR and IP-Western blots. Re-expression of ACVR2 after demethylation with 5-aza-2′-deoxycytidine (5-Aza) was determined. An additional 26 MSS colon cancers were assessed for ACVR2 loss and its mechanism, and ACVR2 loss in all tested cancers correlated with clinicopathological criteria. Results Of 51 MSS colon tumors, 7(14%) lost ACVR2, 2 (4%) ACVR1, and 5(10%) pSMAD2 expression. No somatic ACVR2 mutations were detected. Loss of ACVR2 expression was associated with LOH at ACVR2 (p<0.001) and ACVR2 promoter hypermethylation (p<0.05). ACVR2 LOH, but not promoter hypermethylation, correlated with CIN status. In colon cancer cell lines with fully methylated ACVR2 promoter, loss of ACVR2 mRNA and protein expression was restored with 5-Aza treatment. Loss of ACVR2 was associated with an increase in primary colon cancer volume (p<0.05). Conclusions Only a small percentage of MSS colon cancers lose expression of activin signaling members. ACVR2 loss occurs through LOH and ACVR2 promoter hypermethylation, revealing distinct mechanisms for ACVR2 inactivation in both MSI and MSS subtypes of colon cancer.

BARD1 Expression Predicts Outcome in Colon Cancer

Purpose: BARD1 is a BRCA1-binding partner with tumor suppressive properties. Aberrant splice variants of BARD1 have been detected in various cancers, and it has been postulated that the presence of some splice variants is cancer specific. This is the first study assessing BARD1 expression patterns and correlation with clinical outcome in colon cancer. Experimental Design: We analyzed colon cancer samples for the occurrence of BARD1 splice variants, characterized novel BARD1 splice variants, and quantified the mRNA expression levels of these isoforms in primary colon cancers and their corresponding normal tissue. We tested the correlation of full-length BARD1 protein expression and clinical outcome in primary colon cancer samples. Results: In addition to the full-length BARD1 mRNA, we now find 19 distinct BARD1 splice variants in colon cancer. Contrary to previous assumptions, these splice variants also occur in the adjacent normal colon tissue. Although BARD1 splice variants account for a considerable amount of BARD1 mRNA in both cancer and normal colon samples, distinct variants show a cancer-specific regulation pattern. Consistent with its role as tumor suppressor, we further find that the expression of the full-length BARD1 protein predicts outcome in colon cancer and that loss of full-length BARD1 protein is associated with a poor prognosis (P = 0.0002). Conclusion: Taken together, this is the first report to suggest that BARD1 regulation is an important pathway in colon cancer and that the BARD1 full-length protein may be a useful marker to improve risk stratification in colon cancer patients. Clin Cancer Res; 17(16); 5451–62. ©2011 AACR.

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, BARD1β, results in a more malignant phenotype with decreased RAD51 foci formation, reduced BRCA1 E3 ubiquitin ligase activity, and decreased nuclear BRCA1 protein localization. BARD1β 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 BARD1β 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.

Loss of histone variant macroH2A2 expression associates with progression of anal neoplasm

Aims The macroH2A histone variants are epigenetic marks for inactivated chromatin. In this study, we examined the expression of macroH2A2 in anal neoplasm from anal intraepithelial neoplasia (AIN) to anal squamous cell carcinoma (SCC). Methods AIN and anal SCC samples were analysed for macroH2A2 expression, HIV and human papilloma virus (HPV). The association of macroH2A2 expression with clinical grade, disease recurrence, overall survival and viral involvement was determined. Results macroH2A2 was expressed in normal squamous tissue and lower grade AIN (I and II). Expression was lost in 38% of high-grade AIN (III) and 71% of anal SCC (p=0.002). Patients with AIN with macroH2A2-negative lesions showed earlier recurrence than those with macroH2A2-positive neoplasm (p=0.017). With anal SCC, macroH2A2 loss was more prevalent in the HPV-negative tumours. Conclusions Loss of histone variant macroH2A2 expression is associated with the progression of anal neoplasm and can be used as a prognostic biomarker for high-grade AIN and SCC.

MacroH2A histone variants limit chromatin plasticity through two distinct mechanisms

MacroH2A histone variants suppress tumor progression and act as epigenetic barriers to induced pluripotency. How they impart their influence on chromatin plasticity is not well understood. Here, we analyze how the different domains of macroH2A proteins contribute to chromatin structure and dynamics. By solving the crystal structure of the macrodomain of human macroH2A2 at 1.7 Å, we find that its putative binding pocket exhibits marked structural differences compared with the macroH2A1.1 isoform, rendering macroH2A2 unable to bind ADP‐ribose. Quantitative binding assays show that this specificity is conserved among vertebrate macroH2A isoforms. We further find that macroH2A histones reduce the transient, PARP1‐dependent chromatin relaxation that occurs in living cells upon DNA damage through two distinct mechanisms. First, macroH2A1.1 mediates an isoform‐specific effect through its ability to suppress PARP1 activity. Second, the unstructured linker region exerts an additional repressive effect that is common to all macroH2A proteins. In the absence of DNA damage, the macroH2A linker is also sufficient for rescuing heterochromatin architecture in cells deficient for macroH2A.

Abstract 4020: Characterization of novel BARD1 splice variants in colon cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL BARD1 is a BRCA1 binding partner with putative tumor suppressive properties. Aberrant splice variants of BARD1 have been detected in various cancers, and it has been postulated that the presence of splice variants is cancer-specific. BARD1 expression patterns and their correlation with clinical outcome have not been assessed in colon cancer. In this study, we determined the occurrence and role of BARD1 splice variants in colon cancer. We characterized novel BARD1 splice variants and quantified the mRNA expression levels of BARD1 splice variants and full-length BARD1 in primary colon cancers and their corresponding normal tissue. Further, full-length BARD1 protein expression was correlated with clinical outcome in primary colon cancer samples. Methods: We performed PCR on cDNA from 15 colon cancer samples and matched normal colon tissues using primer targets in the first and last exon of BARD1 mRNA. The PCR produced several bands, that were submitted for direct sequencing. In a second approach the bands were subcloned and subsequently sequenced. The sequences were analyzed and splice variants were characterized. Splice variant specific qPCR primers were designed and expression levels were quantified using optimized SYBR Green qPCR assays. Control assays were performed to assure good RNA/cDNA quality and optimal reaction conditions. In addition, mRNA expression analysis was performed on a cDNA array consisting of pooled cDNA from 16 different human tissues (prostate, heart, brain, testis, placenta, leukocytes, thymus, lung, colon, pancreas, skeletal muscle, liver, kidney, spleen, small intestine, ovary). Full-length BARD1 protein expression was assessed via immunohistochemistry in a separate cohort of 82 primary colon cancer samples. The stained slides were scanned and visualized using the Aperio ImageScope system. Expression levels were assessed and correlated with clinical outcome data. Results: In addition to the full-length BARD1 mRNA, we now find 19 distinct BARD1 splice variants in colon cancer, the majority of which have not been previously described. 18 of the 19 splice variants lack one or more complete exons, thus creating new and specific exon-exon boundaries. Contrary to previous assumptions, these splice variants can also be found in the adjacent normal colon tissue as well as in various other normal human tissues. While BARD1 splice variants account for the majority of BARD1 mRNA, distinct variants show a cancer-specific regulation pattern. Further, loss of full-length BARD1 protein is associated with a poor prognosis (p=0.02). Conclusion: BARD1 splice variants commonly occur in colon cancer and normal colon tissue, with distinct variants showing a cancer-specific expression pattern. Loss of the full-length BARD1 protein correlates with a poor prognosis in colon cancer, consistent with a tumor suppressive function. Taken together, we suggest that splice variant regulation may affect BARD1 function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4020. doi:10.1158/1538-7445.AM2011-4020

Abstract B29: Expression of BARD1 predicts outcome in colon cancer

Background: The BARD1/BRCA1 heterodimer has been implicated in several different pathways crucial for maintaining genomic integrity, DNA repair and cell survival. Complex functions have been ascribed to BARD1: It acts as a chaperone that recruits BRCA1 to the nucleus to form an ubiquitin E3 ligase complex involved in DNA repair and chromosomal stability. On the other hand, BARD1 may also promote cell survival by inhibiting BRCA1-dependent apoptosis through nuclear retention of BRCA1. Mutations in the BARD1 gene, as well as aberrant expression of BARD1 splice isoforms, have been associated with poor prognosis in breast and ovarian cancer. Here, we assess the occurrence and role of BARD1 splice isoforms in colon cancer formation. Methods: Total RNA from primary human colon cancer samples and matched normal colon tissue was extracted and reverse transcribed. Gene specific PCR primers were designed to analyze the occurrence and pattern of BARD1 splice variants in colon cancer. Splice variants were subcloned and sequenced. Protein expression of BRCA1 and BARD1 in paraffin-embedded cancer tissues was detected by immunohistochemistry and correlated with clinicopathologic parameters. Results: mRNA analysis of primary colon cancers confirmed a specific pattern of BARD1 splice isoforms distinct from normal controls. Certain isoforms appear to be tumor specific. Full length BARD1 is upregulated in non-metastatic cancer samples compared to matched normal tissue, which is in line with its anti-apoptotic function. Interestingly, full length BARD1 is dramatically downregulated in metastatic cancer tissue supporting its role as a tumor suppressor. Immunohistochemical analysis of colon cancer samples confirmed the loss of full length BARD1 in metastatic tissues on the protein level and revealed a significant correlation (p Conclusion: Our results suggest critical roles for the BRCA1 chaperone BARD1 in colon cancer formation. Alternative splice variants of BARD1 commonly occur in colon cancer tissue and expression of full-length BARD1 is differentially regulated along the cancer-metastasis axis affecting cancer outcome. We hypothesize that in colon cancer tissue, BRCA1 function critically depends on BARD1 expression. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B29