Tissue Samples Of Cancer Patients Research Articles

The DNA repair pathway as a therapeutic target to synergize with trastuzumab deruxtecan in HER2-targeted antibody–drug conjugate–resistant HER2-overexpressing breast cancer

BackgroundAnti-HER2 therapies, including the HER2 antibody–drug conjugates (ADCs) trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd), have led to improved survival outcomes in patients with HER2-overexpressing (HER2+) metastatic breast cancer. However, intrinsic or acquired resistance to anti-HER2–based therapies remains a clinical challenge in these patients, as there is no standard of care following disease progression. The purpose of this study was to elucidate the mechanisms of resistance to T-DM1 and T-DXd in HER2+ BC patients and preclinical models and identify targets whose inhibition enhances the antitumor activity of T-DXd in HER2-directed ADC-resistant HER2+ breast cancer in vitro and in vivo.MethodsTargeted DNA and whole transcriptome sequencing were performed in breast cancer patient tissue samples to investigate genetic aberrations that arose after anti-HER2 therapy. We generated T-DM1 and T-DXd–resistant HER2+ breast cancer cell lines. To elucidate their resistance mechanisms and to identify potential synergistic kinase targets for enhancing the efficacy of T-DXd, we used fluorescence in situ hybridization, droplet digital PCR, Western blotting, whole-genome sequencing, cDNA microarray, and synthetic lethal kinome RNA interference screening. In addition, cell viability, colony formation, and xenograft assays were used to determine the synergistic antitumor effect of T-DXd combinations.ResultsWe found reduced HER2 expression in patients and amplified DNA repair–related genes in patients after anti-HER2 therapy. Reduced ERBB2 gene amplification in HER2-directed ADC–resistant HER2+ breast cancer cell lines was through DNA damage and epigenetic mechanisms. In HER2-directed ADC–resistant HER2+ breast cancer cell lines, our non-biased RNA interference screening identified the DNA repair pathway as a potential target within the canonical pathways to enhance the efficacy of T-DXd. We validated that the combination of T-DXd with ataxia telangiectasia and Rad3-related inhibitor, elimusertib, led to significant breast cancer cell death in vitro (P < 0.01) and in vivo (P < 0.01) compared to single agents.ConclusionsThe DNA repair pathways contribute to HER2-directed ADC resistance. Our data justify exploring the combination treatment of T-DXd with DNA repair–targeting drugs to treat HER2-directed ADC–resistant HER2+ breast cancer in clinical trials.

Metformin Prevents Tumor Cell Growth and Invasion of Human Hormone Receptor-Positive Breast Cancer (HR+ BC) Cells via FOXA1 Inhibition.

Women with type 2 diabetes (T2D) have a higher risk of being diagnosed with breast cancer and have worse survival than non-diabetic women if they do develop breast cancer. However, more research is needed to elucidate the biological underpinnings of these relationships. Here, we found that forkhead box A1 (FOXA1), a forkhead family transcription factor, and metformin (1,1-dimethylbiguanide hydrochloride), a medication used to treat T2D, may impact hormone-receptor-positive (HR+) breast cancer (BC) tumor cell growth and metastasis. Indeed, fourteen diabetes-associated genes are highly expressed in only three HR+ breast cancer cell lines but not the other subtypes utilizing a 53,805 gene database obtained from NCBI GEO. Among the diabetes-related genes, FOXA1, MTA3, PAK4, FGFR3, and KIF22 were highly expressed in HR+ breast cancer from 4032 breast cancer patient tissue samples using the Breast Cancer Gene Expression Omnibus. Notably, elevated FOXA1 expression correlated with poorer overall survival in patients with estrogen-receptor-positive/progesterone-receptor-positive (ER+/PR+) breast cancer. Furthermore, experiments demonstrated that loss of the FOXA1 gene inhibited tumor proliferation and invasion in vitro using MCF-7 and T47D HR+ breast cancer cell lines. Metformin, an anti-diabetic medication, significantly suppressed tumor cell growth in MCF-7 cells. Additionally, either metformin treatment or FOXA1 gene deletion enhanced tamoxifen-induced tumor growth inhibition in HR+ breast cancer cell lines within an ex vivo three-dimensional (3D) organoid model. Therefore, the diabetes-related medicine metformin and FOXA1 gene inhibition might be a new treatment for patients with HR+ breast cancer when combined with tamoxifen, an endocrine therapy.

Abstract 1331: A novel approach to treating hormonal breast cancer using clinical database and 3D ex vivo model

Abstract Type 2 diabetes (T2D) female patients have a higher risk of being diagnosed and have worse survival for breast cancer compared to non-diabetic females. Although the relationship between these two female diseases has been reported, specific functional studies on which subtypes of breast cancer are affected and which genes are regulated by T2D are little known. Here, I found that Forkhead Box A1 (FOXA1) and Metformin, anti-diabetic medication for T2D, affect hormonal positive (HR+) breast cancer tumor cell growth and metastasis. 14 diabetes-related genes highly expressed in three HR+ breast cancer cell lines but not other breast cancer subtypes using a 53,805 gene database obtained from NCBI GEO. Among the diabetes-related genes, FOXA1, MTA3, PAK4, FGFR3, and KIF22 were highly expressed in HR+ breast cancer from 4,032 breast cancer patient tissue samples using the Breast Cancer Gene Expression Omnibus. Specifically, the high expression of FOXA1 correlated to a worse ER+/PR+ breast cancer patient survival rate. Consistent with this, the loss of FOXA1 inhibited the tumor proliferation and invasion of MCF-7 and T47D HR+ breast cancer cell lines using in vitro. Metformin (1,1-dimethylbiguanide hydrochloride) conspicuously inhibits the tumor cell growth in MCF-7 human hormonal breast cancer cells. Metformin or FOXA1 deletion enhanced Tamoxifen-mediated tumor growth inhibition in HR+ breast cancer cell lines through the ex vivo three-dimensional (3D) organoid model. Therefore, HR+ breast cancer is closely related to T2D, and metformin and FOXA1 inhibition might be an optimal new treatment for patients with HR+ breast cancer when combined with tamoxifen, a hormone therapy. Citation Format: Chaeun (Christine) Song, SeungBaek Lee. A novel approach to treating hormonal breast cancer using clinical database and 3D ex vivo model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1331.

Tumor-infiltrating lymphocytes (TILs) dynamics in breast cancer patients receiving neoadjuvant therapy: A systematic review and meta-analysis.

e12620 Background: Increased baseline tumor-infiltrating lymphocytes (TILs) are associated with improved pathological complete response rates and better prognosis in HER2+ and triple negative breast cancer (TNBC) patients receiving neoadjuvant therapy (NAT). However, the role of TILs dynamics/change (ΔTILs) at the neoadjuvant setting remains unclear, thus a meta-analysis of the published studies was carried out. Methods: Medline, Embase, Cochrane Library and Web of Science Core Collection were searched for studies reporting on TILs expression in paired invasive breast cancer patient tissue samples before and after NAT. Data were extracted by two investigators (Y.Z., E.T.) and discordances were resolved by a third (I.Z.). Outcomes included pooled TILs rates pre- &amp; post-treatment (also per subtype), pooled rates of ΔTILs and direction of change after NAT as well as correlation of ΔTILs with survival outcomes. Heterogeneity was assessed using the I2 statistic. Results: Of 1569 identified entries, 22 studies fulfilled the criteria and provided adequate data for the outcomes of interest. Overall, a significantly decreased level of TILs was observed after NAT in paired samples (pooled OR = 1.60, 95% CI: 1.12-2.30, p = 0.01; TILs as categorical variable). Regarding pooled rates of ΔTILs, a change was observed after NAT, irrespective of BC subtype. Among the different subtypes, the effect of NAT on TILs was most prominent in HER2+ disease with a direction towards decreased TILs to be more common (pooled ΔTILs rates: 14.4% increased vs 46.2%, decreased). In TNBC, bi-directional TIL kinetics were noted (pooled ΔTILs rates: 41.6% increased vs 37.1% decreased). An increase in ΔTILs in TNBC was associated with better disease-free/relapse-free survival in univariate analysis (HR = 0.59, 95% CI: 0.37–0.95, p = 0.03). Substantial between-study heterogeneity was observed in most analyses. Conclusions: The first to our knowledge meta-analysis on TILs dynamics during NAT in BC informs about differences in matched pre- and post-treatment patient samples and the prognostic implications of ΔTILs in TNBC. The potential clinical utility of the longitudinal assessment of immune response during neoadjuvant therapy warrants further investigation in prospective trials.

TRIM27 acts as an oncogene and regulates cell proliferation and metastasis in non-small cell lung cancer through SIX3-β-catenin signaling.

The Wnt/β-catenin pathway plays vital roles in diverse biological processes, including cell differentiation, proliferation, migration, and insulin sensitivity. A recent study reported that the DNA-binding transcriptional factor SIX3 is essential during embryonic development in vertebrates and capable of downregulating target genes of the Wnt/β-catenin pathway in lung cancer, indicating negative regulation of Wnt/β-catenin activation. However, regulation of the SIX3-Wnt/β-catenin pathway axis remains unknown. We measured the expression of TRIM27 and SIX3 as well as investigated whether there was a correlation between them in lung cancer tissue samples. Herein, we found that the E3 ubiquitin ligase, TRIM27, ubiquitinates, and degrades SIX3. TRIM27 induces non-small cell lung cancer (NSCLC) cell proliferation and metastasis, and the expression of β-catenin, S100P, TGFB3, and MMP-9 were significantly inhibited by SIX3. Furthermore, XAV939 is a selective β-catenin-mediated transcription inhibitor that inhibited TRIM27- and SIX3-mediated NSCLC cell proliferation, migration, and invasion. Clinically, lung tissue samples of cancer patients showed increased TRIM27 expression and decreased SIX3 expression. Taken together, these data demonstrate that TRIM27 acts as an oncogene regulating cell proliferation and metastasis in NSCLC through SIX3-β-catenin signaling.

Functional Characterization of Colon-Cancer-Associated Variants in ADAM17 Affecting the Catalytic Domain.

Although extensively investigated, cancer is still one of the most devastating and lethal diseases in the modern world. Among different types, colorectal cancer (CRC) is most prevalent and mortal, making it an important subject of research. The metalloprotease ADAM17 has been implicated in the development of CRC due to its involvement in signaling pathways related to inflammation and cell proliferation. ADAM17 is capable of releasing membrane-bound proteins from the cell surface in a process called shedding. A deficiency of ADAM17 activity has been previously shown to have protective effects against CRC in mice, while an upregulation of ADAM17 activity is suspected to facilitate tumor development. In this study, we characterize ADAM17 variants found in tissue samples of cancer patients in overexpression studies. We here focus on point mutations identified within the catalytic domain of ADAM17 and could show a functional dysregulation of the CRC-associated variants. Since the catalytic domain of ADAM17 is the only region structurally determined by crystallography, we study the effect of each point mutation not only to learn more about the role of ADAM17 in cancer, but also to investigate the structure–function relationships of the metalloprotease.

Abstract P1-06-10: Combined tandem affinity purification mass-spectrometry technique with genome-editing CRISPR-Cas9 knockout screening to identify potential subunits in the BRCA1 complex

Abstract Background: Genomic instability is one of tumor characteristics. Enhancing the DNA damage load or impairing the ability of DNA damage repair has been therapeutics of cancer. Breast cancer type 1 susceptibility gene (BRCA1) has been studied a lot about the DNA damage and repair in breast cancer and ovarian cancer. BRCA1 mutation increases the risk of developing breast cancer by an eighth to tenth. The C terminal domain of BRCA1 can associates with three proteins Abraxas, Bach1 and CtIP in a phosphorylation-dependent manner forming mutually exclusive complexes, namely BRCA1-A, B, and C complexes. The BRCA1-A complex is necessary in DNA damage repair, and study implicates the complex play roles in chemotherapy resistance. Methods: We explored tandem affinity purification mass-spectrometry (TAP-MAS) technique to identify potential subunits associated with NBA1(one component of BRCA1-A complex). Then we made the genome-editing CRISPR-Cas9 sgRNA library into lentivirus to infect U2OS cells. And 5 Gy dose of Ionizing radiation (IR) was used to induce DNA damage on the cell. After 14 days cultivation, we extracted the DNA from the cells, performed polymerase chain reaction (PCR) and analyzed the correlation between potential genes and DNA damage-repair passage by bioinformatics methods. We generated 200 breast cancer patient tissue samples in our cancer center and performed immunostaining assay. Results: By the TAP-MAS technique of NBA1 tagged with HA and Flag, we found 93 potential subunits except those known subunits in BRCA1-A complex. Combined with CRISPR-Cas9 sgRNA library screening, we scored the relativity of DNA damage and repair passageway of identified 93 potential subunits. We found that nucleoside-triphosphatase, cancer-related (NTPCR) as a new potential subunit of BRCA1-A complex (P value=0.0034) had the highest score. Endogenous and exogenous co-IP validated NTPCR physically associates with subunits within BRCA1-A complex. Besides, we found that NTPCR associated with the same domain of NBA1 as the other subunits in BRCA1-A complex. Immunohistochemistry of patient tissue samples indicated that high levels of NTPCR expression was correlated with poor prognosis in multivariate analysis (HR: 4.990; 95%CI: 1.433-17.378; p value: 0.012). Conclusion: NTPCR is a new subunit in BRCA1-A complex. And high expression of NTPCR is a negative prognostic factor in breast cancer patients. Citation Format: Zhao H, Jiang H, Sun W, Shao Z, Hu X. Combined tandem affinity purification mass-spectrometry technique with genome-editing CRISPR-Cas9 knockout screening to identify potential subunits in the BRCA1 complex [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 P1-06-10.

Silencing ROR1 and ROR2 inhibits invasion and adhesion in an organotypic model of ovarian cancer metastasis.

OBJECTIVEElevated expression of the ROR1 and ROR2 Wnt receptors has been noted in both the tumour and stromal compartments of ovarian cancer patient tissue samples. In vitro studies have suggested these receptors play a role in ovarian cancer metastasis. However, these previous studies have utilised simple 2D in vitro models to investigate cancer cell growth and migration, which does not allow investigation of stromal involvement in Wnt driven metastasis.AIMTo investigate targeting ROR1 and ROR2 using a primary co-culture 3D model of epithelial ovarian cancer dissemination to the omentum.METHODSPrimary fibroblasts (NOF) and mesothelial (HPMC) cells were isolated from fresh samples of omentum collected from women with benign or non-metastatic conditions and cultured with collagen to produce a organotypic 3D model. Stable shRNA knockdown of ROR1, ROR2 and double ROR1/ROR2 in OVCAR4 cells were plated onto the 3D model to measure adhesion, or using a transwell to measure invasion. Gene expression changes in primary cells upon OVCAR4 interaction was evaluated using indirect transwell co-culture.RESULTSDouble knockdown of ROR1 and ROR2 strongly inhibited cell adhesion (p<0.05) and invasion (P<0.05) to the omentum model. ROR2 was up regulated in primary fibroblasts when cultured with OVCAR4 (P=0.05) and ectopic overexpression of ROR2 in NOFs inhibited cell proliferation (P<0.01) but increased cell migration.CONCLUSIONThe combination of ROR1 and ROR2 signalling influences ovarian cancer dissemination to the omentum, however ROR2 may also play a role in stromal activation during metastasis. Therefore, targeting both ROR1 and ROR2 may be a powerful approach to treating ovarian cancer.

MAEL expression links epithelial-mesenchymal transition and stem cell properties in colorectal cancer.

MAEL plays a central role during spermatogenesis by repressing transposable elements and preventing their mobilisation, however, its role on cancers is unclear. In this study, MAEL expression was analysed in a tissue microarray containing 185 samples of primary colon cancer tumor samples and human colon cancer cell lines. The effect of MAEL on cell proliferation, tumorigenesis, metastasis and drug resistance was examined in vitro and in vivo. Immunoprecipitation assay, confocal immunofluorescent analysis and luciferase assay were used for mechanism study. As results, MAEL was significantly upregulated in colon cancer patient tissue samples, and elevated MAEL protein levels positively correlated with overall survival and disease free survival of colon cancer patients. Using in vitro and in vivo models, we demonstrated that MAEL expression was correlated with cell proliferation, invasion and drug resistance of colon cancer cells by inducing epithelial-mesenchymal transition and stemness characteristics. Mechanistically, our study demonstrated that MAEL interacts with Snail and inhibit E-cadherin promoter activity. Collectively, MAEL is an oncogene that plays an important role in the development and progression of colon cancer, which may be a novel potential therapeutic target for colon cancer.

Abstract 1265: LAMP2 overexpression in the plasma membrane of breast cancer cells in response of chronic acidosis as a new imaging and therapeutic target

Abstract A combination of poor vasculature perfusion, hypoxia, and increased flux of carbons through fermentative glycolysis lead to extracellular acidosis in solid tumors; with extracellular pH values as low as 6.5. The proton concentration increases within the lumen due to diffusion limitations and increased production of acid from hypoxic-glycolytic cells, causing the interior of the lumen to become highly acidic. The glycolytic phenotype can become “hardwired” as Warburg proposed, leading to the continued generation of metabolic acids even in well-oxygenated conditions. This acidified habitat is constant and imparts a Darwinian selection pressure that favors cells that have adapted mechanisms to be resistant to acid mediated apoptosis, and this likely increases with increasing cancer stage. In order to understand tumor acidity, we investigated protein profiles of acid-adapted and non-adapted breast cancer cells (MCF-7) using SILAC-proteomics followed by LC-MRM confirmation. A large number of lysosomal proteins were observed to be upregulated in acid-adapted cells and, among these was the lysosome associated membrane protein 2 (LAMP2) confirmed by MRM. This protein is of particular interest because it is heavily glycosylated and functions to protect lysosomal membrane from acid proteolysis. Using independent in vitro as well as in vivo approaches such as intravital fluorescent microscopy of dorsal window chamber mouse xenografts, we observed that LAMP2 was associated with acid-adaptation. In vivo, treatment of animals with bicarbonate to reduce tumor acidity led to reductions in LAMP2. Further, mRNA-expression profiling (microarray) of patient tumor samples and IHC of Tissue Microarray (TMA) of breast cancer patient tissue samples, revealed a high correlation of LAMP2 expression with progression and metastasis of breast tumors. We also applied LAMP2 staining on the whole breast tumor samples of different stages and observed that LAMP2 was associated with the peri-luminal regions of DCIS, as well as regions of local invasion, which are expected to be acidic. Finally, we observed both in tumors by IHC and subsequently in acid adapted cells by ICC that, while LAMP2 was localized in lysosomes and late endosomes under normal pH conditions, it was redistributed to the plasma membrane under acidic pH conditions. To further investigate this cell membrane localization we carried out western blot on membrane extract of acid adapted cells. Based on these observations, we propose a novel cell defense mechanism wherein cells chronically exposed to an acidic environment translocate LAMP2 to their surface, which protects the plasmalemma via an unstirred layer effect. To prove the role of LAMP2 on acid adaptation we did shRNA and siRNA knockdown followed by in vitro and in vivo acid exposure of the cells. We further propose that this pathway may provide a novel therapeutic target against breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Mehdi Damaghi, Robert Sprung, Narges Tafreshi, Veronica Estrella, John Koomen, David Morse, Robert Gillies. LAMP2 overexpression in the plasma membrane of breast cancer cells in response of chronic acidosis as a new imaging and therapeutic target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1265. doi:10.1158/1538-7445.AM2015-1265

Abstract 2338: Histone demethylase KDM3A: Epigenetic target for pancreatic ductal adenocarcinoma (PDAC)

Abstract Purpose: A growing body of evidence suggests that cancer stem cells/tumor initiating cells (CSC/TIC) within a solid tumor including in pancreatic cancer initiate and sustain tumor growth. Our preliminary data implicates KDM3A (histone lysine demethylase 3A)/Jmjd1A (jumonji domain demethylase 1A), non-heme iron (II)- α-ketoglutarate (kg) dependent enzyme, as a prime regulator of stems cell self-renewability for cancer progression and is overexpressed in PDAC. Hypoxia most prominently controls malignant properties of cancer cells by stimulating hypoxia inducible factor 1 (HIF1). The fact that HIF1 binds to its responsible elements (HREs) in the KDM3A promoters and subsequently up-regulates its expression could explain why hypoxia exacerbates malignancy. Based on these observations, we hypothesize that hypoxia induces KDM3A that regulates self-renewability of CSC/TIC, thereby promoting tumor initiation. Experimental Procedure: We have used RNA technology to silence KDM3A in different pancreatic cancer cells and overexpressed KDM3A in non-cancerous immortal pancreatic ductal epithelial cells (HPNE). We have determined KDM3A expression in pancreatic cancer patient's samples. We performed quantitative chromatin immunoprecipitation (ChIP) analysis as an indicator of KDM3A recruitment. Results: KDM3A was overexpressed in human pancreatic cancer patient tissue samples particularly in the area of the pancreatic ducts and also in pancreatic cancer cells. Knockdown of KDM3A in pancreatic cancer cells significantly reduced the enzyme activity resulting in increased substrate (H3K9Me2) expression. KDM3A knockdown cells decreased oncogenic potential as demonstrated by inhibition of proliferation, clonogenicity, sphere-forming potential, migration and tumor formation in orthotopic mouse model (immunocompromised mice). KDM3A overexpressed transformed HPNE cells increased oncogenic potential as evidenced by foci formation, pancosphere formation and tumor formation in orthotopic mouse model. ChIP following deep sequencing analysis suggested that KDM3A recruited one of the CSC stem cell markers, DCLK1. N-oxaloglycine, α-kg inhibitor, inhibited KDM3A enzyme activity and spheroid formation in overexpressing HPNE cells and in pancreatic cancer cells.Conclusions: KDM3A was overexpressed in PDAC. ChIP-seq analysis revealed that KDM3A recruitment was higher when DCLK1 is expressed. Taken together, KDM3A appears to be responsible, at least in part, for PDAC progression and could be a novel preventive or therapeutic target in pancreatic cancer. Citation Format: Satheesh K. Sainathan, Santanu Paul, Kanagaraj Palaniyandi, Dharmalingam Subramaniam, Satish Ramalingam, Ossama W. Tawfik, Tomoo Iwakuma, Danny R. Welch, Subhash B. Padhye, Shrikant Anant, Animesh Dhar. Histone demethylase KDM3A: Epigenetic target for pancreatic ductal adenocarcinoma (PDAC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2338. doi:10.1158/1538-7445.AM2015-2338

Abstract P2-06-09: Inflammation promotes stem-like characteristics and increases stemness of breast epithelial cells

Abstract Triple negative breast cancer (TNBC) accounts for approximately 15% of all breast cancers and is more likely to affect younger women, women of African American descent, and BRCA1 mutation carriers. TNBC is typically an aggressive cancer and is defined as the absence of positive staining for estrogen (ER) and progesterone (PR) receptor, and a lack of amplification of HER2. Due to its receptor status, TNBC is insensitive to the conventional targeted treatment utilized in ER/PR/HER2 positive breast cancer leading to poor prognosis and early visceral metastasis with survival rates for women who relapse within 5 years of treatment being significantly lower than hormone receptor positive breast cancer. Therefore, understanding the mechanisms that drive the formation of TNBC can aid in determining targets to develop better treatment options. We investigated differences in the tumor microenvironment between TNBC and ER positive breast cancer patient tissue samples. Hematoxylin &amp; eosin stain of invasive ductal carcinomas revealed that TNBC patients had higher amounts of tumor infiltrating leukocytes compared to ER positive breast cancers. In premalignant breast cancer patients, ER negative ductal carcinoma in situ (DCIS) patients had significantly higher infiltrating leukocytes compared to ER positive DCIS indicating that inflammation may be a contributing factor in TNBC. Research on molecular profiling of TNBC has revealed that a subset of gene expression patterns are associated with the basal-myoepithelial cells rather than the luminal cells, however, previous evidence indicates that TNBC arises from luminal progenitors which undergo an abnormal conversion to basal-like progenitors with enhanced growth and survival promoting characteristics. We propose this conversion occurs due to the presence of inflammatory signals early in tumor development which trigger a cascade of events that leads to acquisition of a basal-like phenotype in luminal progenitor cells and upregulation of stem-like properties, resulting in the formation of basal-like breast cancer/TNBC. Treatment of non-malignant breast epithelial cells with IL-6 increased sphere-forming efficiency under stem cell growth promoting conditions and an inhibitor of the IL-6/STAT3 pathway decreased ALDH enzymatic activity as measured by Aldefluor assay. Also, treatment with IL-6 downregulated the luminal marker, epithelial specific antigen (ESA), and increased the expression of the myoepithelial marker, CD49f, on the cell surface of non-malignant breast epithelial cells, indicating an increase in the basal-like cell population and a loss of the luminal cell population. Therefore, inflammation appears to be a common feature in TNBC compared to ER positive breast cancer and the presence of an inflammatory stimulus in the breast may promote the conversion of luminal to basal-like cells, promoting the upregulation of stem-like properties and development of TNBC. These data suggest that anti-inflammatory drugs could potentially be used to inhibit these events from occurring in the development of TNBC, leading to decreased tumor growth and better treatment options. Citation Format: Jennifer Sims-Mourtada, Kimberly M Arnold, Lynn Opdenaker, Daniel Flynn. Inflammation promotes stem-like characteristics and increases stemness of breast epithelial cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-09.

GHSR hypermethylation: a promising pan-cancer marker

The advent of high-throughput technologies such as microarrays, global gene knockout, and next generation sequencing (NGS) has revolutionized the field of molecular oncology. Large-scale endeavors of the kind represented by the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) are generating comprehensive portraits of human cancers at different molecular levels spanning over genome, epigenome and transcriptome landscapes. These data have made substantial contributions to our knowledge of the molecular biology of cancer, and have highlighted novel factors which had not been acknowledged previously. For example, emerging data indicate that the mutational makeup of tumors can significantly vary based on geographical variations among patients,1 pointing to the contribution of potentially different mechanisms (e.g. environmental) to tumorigenesis in different populations. On the other hand, cancer profiling data have also provided catalogues of cancer-associated molecular variations, which can serve as reliable biomarkers for cancer prevention or personalized management of patients. Successful examples include the refined diagnosis of disease subtype in breast cancer using gene expression signatures and protein markers, and stratifying patients affected by colorectal cancer for anti-EGFR therapy based on KRAS mutations. In recent years, much attention has been focused on epigenetic marks, in particular on the development of DNA methylation-based markers. Abnormal DNA methylation patterns are uniquely attractive for marker development: (i) they are abundant in tumors; (ii) they happen at early stages of tumorigenesis; and (iii) as covalently bound modifications to DNA molecules, they are stable in different body fluids. These characteristics point to the notion that monitoring the presence and abundance of methylated DNA in “liquid biopsy” samples from cancer patients or people who are at high risk for cancer can facilitate non-invasive cancer prognosis and early diagnosis. Indeed, recent studies have reported successful examples of assaying cancer-associated DNA methylation marks in body fluids in order to monitor response to treatment2 or tumor recurrence3 in different cancers. Likewise, new data have shown that DNA methylation markers perform as well as routine clinical procedure (e.g. cytology examination) in the prioritization of patients for clinical management.4 These reports together with several other recent studies have demonstrated that tools and technologies for reliable screening of abnormal DNA methylation patterns in non-invasive manners have become available. An essential step in this respect, however, is the identification of truly informative markers that are to be interrogated in screening procedures. Accordingly, it is of paramount importance to factor for changes in epigenetic patterns that happen naturally during life-time when looking for sensible cancer markers.5 In an earlier study, we had found DNA hypermethylation in the promoter of the growth hormone secretagouge receptor (GHSR) gene as an epigenetic marker of highest accuracy for detection of breast cancer.6 This finding was supported by analyzing tumors as well as an extensive list of control samples including normal-appearing tissue samples from cancer patients and healthy individuals, as well as benign lesions. The gene promoter was significantly hypermethylated in both invasive and non-invasive in situ ductal breast cancer as compared to other samples. Moreover, we noted that GHSR hypermethylation may be involved in an epigenetic field defect in breast cancer, as the level of methylation was higher in normal-appearing tissue samples of cancer patients compared to those from healthy individuals after adjustment for age. These findings showed that while GHSR hypermethylation is a cancer specific pattern, it is detectable in early stage tumors. Motivated by the high accuracy of GHSR hypermethylation for detection of breast cancer (with a sensitivity and specificity of 89.3 and 100%, respectively), we expanded our study to other cancers elucidating if this pattern could represent a pan-cancer marker. We observed that GHSR hypermethylation is able to discriminate cancers of lung, breast, prostate, pancreas, colorectum as well as B-cell chronic lymphocytic leukemia from tissue-matched control samples.7 Similar to the abovementioned cancers, the locus was also hypermethylated in glioblastoma samples. By including tumors of different stages, where available, we showed that GHSR hypermethylation is detected already in early-stage tumors. That is, while all tumors were significantly hypermethylated compared to non-tumoral samples, no significant differences existed for this marker between tumors of lower and higher stages. Collectively, these data indicate that GHSR hypermethylation is a pan-cancer marker regardless of the tissue from which the tumor originates. Therefore, interrogating GHSR methylation may provide a supplemental approach to routine clinical examination for cancer diagnosis. Analyzing this marker in plasma samples and other body fluid from cancer patients will be the next step in verifying its usefulness in non-invasive screening procedures. Mechanistically, it is plausible to assume that GHSR hypermethylation may have a functional role in tumorigenesis in different cancers or there is an inherent susceptibility of the locus to DNA methylation, which is acquired by accelerated cell proliferation. Future studies should address these possibilities.

Overexpression of EGFR protein in Bruneian lung cancer patients.

Lung cancer is the leading cause of cancer death in Brunei Darussalam, accounting for almost 20% of the total. The epidermal growth factor receptor (EGFR) is a member of the erbB family of tyrosine kinase receptor proteins, which includes c-erbb2(HER2/neu), erb-B3, and erb-B4. EGFR overexpression is found in a third of all epithelial cancers, often associated with a poor prognosis. Protein expression of EGFR in 27 cases of lung cancer tissue samples and 9 cases of normal lung tissue samples was evaluated using an immunohistochemical approach. The results demonstrated significant increase and overexpression of EGFR in Bruneian lung cancer tissue samples in comparison to normal lung tissue. However, there was no significant relationship between clinicopathologic variables (age and sex) of patients and EGFR protein expression. EGFR is overexpressed in Bruneian lung cancer patient tissue samples in comparison to normal lung tissue samples. This may indicate that EGFR protein over expression plays an important role in the genesis of this type of cancer in Brunei Darussalam.

LSD1 sustains pancreatic cancer growth via maintaining HIF1α-dependent glycolytic process

The histone demethylase LSD1 (lysine specific demethylase 1) plays an important role in the epigenetic regulation of gene transcription. Our study investigated the role of LSD1 in pancreatic cancer and demonstrated that LSD1 was significantly up-regulated in pancreatic cancer patient tissue samples, and elevated LSD1 protein levels positively correlated with overall survival of pancreatic cancer patients. Using in vitro and in vivo models, we demonstrated that knock-down of LSD1 repressed proliferation and tumorigenicity of pancreatic cancer cells. Mechanistically, our study demonstrated that LSD1 synergized with HIF1α (hypoxia inducible factor-1α) in maintaining glycolytic process, which fueled pancreatic cancer uncontrolled proliferation.

Abstract 3216: Using protein microarray technology to develop mono-specific anti-ERCC1 mAbs for anatomic pathology applications.

Abstract Antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is a predictive biomarker for cisplatin based chemotherapy. High ERCC1 expression is linked to drug resistance to cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissue samples. By using high density protein microarray chip, we discovered that 8F1 not only reacts with its authentic target ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity to PCYT1A was further confirmed by other immunoassays, including Western blot, antigen absorption test, and immunohistochemistry (IHC) analyses. In this study, we also discovered that PCYT1A gene expression level is even significantly higher than ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. In summary, 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on protein microarray chip test and both worked in IHC application. Citation Format: Donghui Ma, Youmin Shu, Kehu Yuan, Wei-Wu He. Using protein microarray technology to develop mono-specific anti-ERCC1 mAbs for anatomic pathology applications. [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 3216. doi:10.1158/1538-7445.AM2013-3216

Using protein microarray technology to screen anti-ERCC1 monoclonal antibodies for specificity and applications in pathology

BackgroundAn antibody with cross-reactivity can create unexpected side effects or false diagnostic reports if used for clinical purposes. ERCC1 is being explored as a predictive diagnostic biomarker for cisplatin-based chemotherapy. High ERCC1 expression is linked to drug resistance on cisplatin-based chemotherapy. 8F1 is one of the most commonly used monoclonal antibodies for evaluating ERCC1 expression levels in lung cancer patient tissues, but it has been noted that this antibody cross-reacts with an unknown protein.ResultsBy using a high density protein microarray chip technology, we discovered that 8F1 not only reacts with its authentic target, ERCC1, but also cross-reacts with an unrelated nuclear membrane protein, PCYT1A. The cross-reactivity is due to a common epitope presented on these two unrelated proteins. Similar to the subcellular localization of ERCC1, IHC tests demonstrated that PCYT1A is localized mainly on nuclear membrane. In this study, we also discovered that the PCYT1A gene expression level is significantly higher than the ERCC1 gene expression level in a certain population of lung cancer patient tissue samples. To develop the best monoclonal antibody for ERCC1 IHC analysis, 18 monoclonal antibodies were generated and 6 of them were screened against our protein microarray chip. Two clones showed high mono-specificity on the protein microarray chip test and both worked for the IHC application.ConclusionIn summary, the 8F1 clone is not suitable for ERCC1 IHC assay due to its cross-reactivity with PCYT1A protein. Two newly generated monoclonal antibodies, 4F9 and 2E12, demonstrated ultra-specificity against ERCC1 protein and superior performance for IHC analyses.

Analysis of the Human Cancer Glycome Identifies a Novel Group of Tumor-AssociatedN-Acetylglucosamine Glycan Antigens

The cell surface is covered by a dense layer of protein- and lipid-linked glycans. Although it has been known that distinct glycan structures are associated with cancer, the whole spectrum of cancer-associated glycans has remained undiscovered. In the present study, we analyzed the protein-linked cancer glycome by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric glycan profiling of cancer patient tissue samples. In lung cancer, we detected accumulation of a novel group of tumor-associated glycans. These protein-linked glycans carried abnormal nonreducing terminal beta-N-acetyl-D-glucosamine (GlcNAc) residues. A similar phenomenon was also detected in structural analyses of tumor-derived glycosphingolipids. This showed that glycan biosynthesis may dramatically change in cancer and that direct glycome analysis can detect the resulting marker glycans. Based on the structural knowledge, we further devised a covalent labeling technique for the detection of GlcNAc-expressing tumors with a specific transferase enzyme. In normal tissues, terminal GlcNAc antigens are capped by galactosylation. Similarly to common cancer-associated glycan antigens T, Tn, and sialyl-Tn, the newly discovered GlcNAc antigens result from incomplete glycosylation. In conclusion, the identified terminal GlcNAc glycans should be recognized as a novel class of tumor markers.