FOLR1 Expression Research Articles

Abstract 2358: KHK2805, a novel ADCC- and CDC-enhanced anti-FOLR1 antibody with AccretaMab® technology, shows a potent anti-tumor activity in combination with pemetrexed

Abstract Introduction: Folate receptor 1 (FOLR1, FR alpha) is a folate transporter which is expressed in many cancers including ovarian cancer (OvC) and non-small cell lung cancer (NSCLC), and which is an attractive target for cancer therapy, is currently the subject of ongoing studies. We established KHK2805, a novel anti-FOLR1 monoclonal antibody with AccretaMab® technology to enhance both the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activities. It was demonstrated that KHK2805 exhibits markedly high ADCC and CDC activity levels in clinical samples from ovarian cancer patients, while a tolerable safety profile has been observed in preclinical models using cynomolgus monkeys (100 mg/kg weekly for 4 weeks, intravenously). A greater understanding of the biology of FOLR1 is important for providing a novel therapeutic option for KHK2805 for patients with cancer. Pemetrexed (PEM), a second-generation anti-folate which inhibits thymidylate synthase, glycinamide ribonucleotide transformylase and dihydrofolate reductase, is used as a standard therapy for patients with cancers such as NSCLC. It is not fully understood how PEM treatment affects the expression of FOLR1 on cancer cells. We therefore examined the level of FOLR1 expression in cancer cells after PEM treatment, and the anti-tumor activity of KHK2805 in combination with PEM. Materials and Methods: The FOLR1 expression levels after PEM treatment were examined in OvC, NSCLC, and endometrial cancer cells by flow cytometry. The ADCC activity of KHK2805 against PEM-treated cells was evaluated. The anti-tumor activity of KHK2805 in combination with PEM was investigated in SCID mice. Results: Flow cytometry showed that PEM treatment increased the FOLR1 expression of various cancers such as OvC (IGROV1, SKOV3, MCAS), NSCLC (NCI-H1437, NCI-H2228), and endometrial cancer (MESSA, HEC1A, HEC1B) cells. In addition, PEM treatment enhanced the ADCC activity of KHK2805 against MCAS, NCI-H1437, and NCI-H2228, in comparison to cells that did not receive PEM treatment. Furthermore, the anti-tumor activity of KHK2805 in SCID mice bearing subcutaneous MCAS tumors was enhanced by PEM treatment. Conclusions: PEM induced further FOLR1 expression in OvC, NSCLC, and endometrial cancer, resulting in the enhancement of the ADCC activity of KHK2805. The use of KHK2805 with PEM might therefore be a potent therapeutic option. Citation Format: Munetoshi Ando, Keiko Nagata, Toshihiko Ishii, Ryuichiro Nakai, Takeshi Takahashi. KHK2805, a novel ADCC- and CDC-enhanced anti-FOLR1 antibody with AccretaMab® technology, shows a potent anti-tumor activity in combination with pemetrexed. [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 2358.

Abstract C124: A novel anti-FOLR1 antibody developed with AccretaMab® technology, KHK2805, exhibits potent anti-cancer activity against ovarian cancer samples with the FOLR1 expression

Abstract Introduction: Folate receptor alpha (FOLR1) is a folate transporter expressed in many cancers, including ovarian cancer. Currently, several clinical trials of FOLR1-targeting drugs [conventional IgG1 antibodies, which exhibit antibody-dependent cellular cytotoxicity/complement dependent cytotoxicity (ADCC/CDC) activities, folic acid or antibody-drug conjugates and vaccines] have been conducted for ovarian and lung cancer. Therefore, FOLR1 is a remarkable target for cancer therapy under ongoing investigation. We established KHK2805, a novel anti-FOLR1 monoclonal antibody, using AccretaMab® technology to enhance both ADCC and CDC activities. Translational research (TR) using clinical samples is essential for determining whether a novel drug shows potent efficacy in clinical studies. In this study, we evaluated the anti-cancer activity of KHK2805 using malignant ascites and serum samples from patients with ovarian cancer. In addition, the FOLR1 expression was evaluated immunohistochemically using ovarian cancer tissues. Materials and Methods: An autologous ADCC assay was conducted using cells from the malignant ascites of ovarian cancer patients, in which both malignant cells (target cells) and immune cells (effector cells) were present. Similarly, the CDC activity was evaluated using supernatant of the malignant ascites obtained from the patients. Furthermore, a CDC assay using the serum of ovarian cancer patients was conducted. An immunohistochemical protocol was established using KM4193, the parental rat antibody of KHK2805, and formalin-fixed, paraffin-embedded ovarian cancer samples were immunohistochemically stained with KM4193. Results: KHK2805 showed potent ADCC activity against FOLR1-positive ovarian cancer cells in the autologous setting using the malignant ascites samples of the ovarian cancer patients, showing a clearly higher activity than that of the conventional anti-FOLR1 antibody. In addition, the CDC activity of KHK2805 was higher than that of the conventional anti-FOLR1 antibody under conditions using the supernatant of malignant ascites or serum from the ovarian cancer patients. Therefore, KHK2805 is thought to have markedly higher killing activity against tumor cells in patients with ovarian cancer. An immunohistochemical examination of the FOLR1 expression showed that the ovarian cancer tissues were positively stained with KM4193. Conclusions: TR using clinical samples from patients with ovarian cancer demonstrated that KHK2805 may be a promising novel anti-FOLR1 ovarian therapeutic agent with a potent antitumor activity. Citation Format: Kaito Nihira, Munetoshi Ando, Keiko Nagata, Maiko Adachi, Yui Suzuki, Yutaka Kanda, Takeshi Oshima, Ken-ichiro Nan-ya, Masanori Hiura, Toshihiko Ishii, Ryuichiro Nakai, Takeshi Takahashi. A novel anti-FOLR1 antibody developed with AccretaMab® technology, KHK2805, exhibits potent anti-cancer activity against ovarian cancer samples with the FOLR1 expression. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C124.

Abstract C123: A novel anti-FOLR1 antibody developed with AccretaMab® technology, KHK2805, exhibits markedly high ADCC/CDC activity and a tolerable safety profile in preclinical models

Abstract Introduction: Folate receptor alpha (FOLR1) is a member of the folate transporter family expressed on normal tissues and overexpressed in multiple types of tumors, such as ovarian cancer, uterine cancer, non-small cell lung cancer, gastric cancer, breast cancer and kidney cancer. Currently, several clinical trials of FOLR1-targeting drugs [conventional IgG1 antibodies, which exhibit antibody-dependent cellular cytotoxicity/complement-dependent cytotoxicity (ADCC/CDC) activities, folic acid or antibody-drug conjugates and vaccines] have been conducted for ovarian and lung cancer. Therefore, FOLR1 is a remarkable target for cancer therapy under ongoing investigation. AccretaMab® technology involves combining both the POTELLIGENT®, a clinically validated ADCC-enhanced technology, and COMPLEGENT®, a new CDC-enhanced technology, systems to result in a superior technology for enhancing the killing activity of antibodies. KHK2805 is a novel humanized and CDR-altered anti-FOLR1 antibody developed with AccretaMab® technology. In this study, we evaluated the anti-cancer activity of KHK2805 in preclinical ovarian cancer models, both in vitro and in vivo, and confirmed the safety profile of KHK2805 in cynomolgus monkeys, since KHK2805 cross-reacts to cynomolgus monkey FOLR1. Materials and Methods: The binding kinetics of KHK2805 against recombinant FOLR1 (rFOLR1) were measured using the Biacore system. The epitope was determined with an ELISA against rFOLR1s. The in vitro ADCC and CDC activities against FOLR1-positive ovarian cancer cells were evaluated using PBMCs and serum from healthy volunteers. The in vivo anti-tumor activity of KHK2805 was examined using a SCID mouse model. The safety profile of KHK2805 was evaluated in cynomolgus monkeys. Results: KHK2805 induced potent ADCC and CDC activities against FOLR1-positive ovarian cancer cells. The ADCC activity of KHK2805 was significantly higher than that of the conventional anti-FOLR1 antibody. Furthermore, KHK2805 showed a potent ADCC activity against ovarian cancer cells with a low FOLR1 expression or low folic acid-uptake activity, which may be difficult to target with current FOLR1-targeting drugs. The results also showed that the markedly higher ADCC activity of KHK2805 was caused by its super-high affinity, unique epitope and use of AccretaMab® technology. In addition, the CDC activity of KHK2805 was also clearly higher than that of the conventional anti-FOLR1 antibody. This indicates that the higher CDC activity of KHK2805 is due to the application of protein engineering of CDR alterations and AccretaMab® technology. Moreover, the potent anti-tumor activity of KHK2805 was observed in a peritoneal dissemination model in SCID mice. Finally, we completed preliminary safety experiments with KHK2805. A repeated-dose toxicity study of KHK2805 (weekly 100 mg/kg for 4 weeks, intravenously) showed an acceptable tolerability profile in cynomolgus monkeys. Conclusions: KHK2805 may be a promising novel anti-FOLR1 therapeutic agent with a potent anti-tumor activity and tolerable safety profile for patients with the FOLR1 expression. Citation Format: Munetoshi Ando, Keiko Nagata, Hiroshi Ando, Mariko Nakano, Naoya Kameyama, Tsuguo Kubota, Maiko Adachi, Yui Suzuki, Kazuyasu Nakamura, Toshihiko Ishii, Ryuichiro Nakai, Takeshi Takahashi. A novel anti-FOLR1 antibody developed with AccretaMab® technology, KHK2805, exhibits markedly high ADCC/CDC activity and a tolerable safety profile in preclinical models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C123.

Abstract 5132: Evaluation of multi-marker in tissue-derived cancer cells from patients with epithelial ovarian carcinoma

Abstract Objective: The objective of this study is to refine a simple, rapid and reproducible method for the isolation and characterization of epithelial ovarian cancer (EOC) cells from ovarian tumor tissues and to evaluate the intracellular expression of CK-7, CA-125, HE4 and FOLR-1 in primary ovarian cancer cells. Methods: Fourteen patients diagnosed with papillary serous adenocarcinoma of ovary were selected in ASAN Medical Center. Isolation of tumor EOC cells involves the mechanical disruption of the tumor tissue and placed directly into culture with very little manipulation. Primary ovarian cancer cells were monitored after initial plating and every day for following 15 days. Confocal laser scanning microscopy was used for the imaging of intracellular immunofluorescence of CK-7, CA-125, HE4 and FOLR-1. Results: Two days after plating primary ovarian cancer cells began to adhere to the plate by the presence of small adherent clusters. The primary ovarian cancer cells became a confluent monolayer and displayed the typical epithelial cobblestone morphology. The mean duration of passage was 8.5 days, it was revealed variously in the primary ovarian cancer cells. Immunofluorescent detection of CK-7 expression in EOC cells demonstrates the epithelial origin, however their expression was not constant. CA-125, HE-4 and FOLR-1 were expressed by all tissue-derived EOC cells. The expression pattern of CA-125, HE-4 and FOLR-1 was different in each patient and that was not correlated with the serum level of CA-125. Conclusions: The isolation and characterization of tissue-derived cancer cells from patients with EOC using the expression of CK-7, CA-125, HE4 and FOLR-1 were reasonable method. This result could provide clinically relevant models suitable for investigation of the ex vivo biological characteristics of ovarian cancers. Citation Format: Shin-Wha Lee, Sang-Eun Lee, Ha-Young Lee, Dae-Yeon Kim, Jong-Hyeok Kim, Yong-Man Kim, Young-Tak Kim, Joo-Hyun Nam. Evaluation of multi-marker in tissue-derived cancer cells from patients with epithelial ovarian carcinoma. [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 5132. doi:10.1158/1538-7445.AM2015-5132

Expression of folate receptors alpha and beta in normal and cancerous gynecologic tissues: correlation of expression of the beta isoform with macrophage markers.

BackgroundFolate receptor alpha (FOLR1/FRA) is expressed in a number of epithelial cancers and in particular epithelial ovarian cancer (EOC), especially of the serous histotype. Recent studies have shown that EOC originates from the fallopian tube fimbriae rather than from epithelial cells lining the ovary. We have previously shown by immunohistochemistry a strong correlation between FRA expression in EOC and normal and fallopian adenocarcinoma. Folate receptor beta (FOLR2/FRB) has been described to be expressed by macrophages both in inflammatory disorders and certain epithelial cancers. Given the high sequence identity of these two folate receptor family members we sought to investigate the architectural and cell-specific expression of these two receptors in gynecologic tissues.MethodsRNA scope, a novel chromogenic in situ hybridization assay tool, was used to examine expression of the alpha (FOLR1) and beta (FOLR2) isoforms of folate receptor relative to each other as well as to the macrophage markers CD11b and CD68, in samples of normal fallopian tube and fallopian adenocarcinoma as well as normal ovary and EOC.ResultsWe demonstrated expression of both FOLR1 and FOLR2 in EOC, normal fallopian tube and fallopian adenocarcinoma tissue while very little expression of either marker was observed in normal ovary. Furthermore, FOLR2 was shown to be expressed almost exclusively in macrophages, of both the M1 and M2 lineages, as determined by co-expression of CD11b and/or CD68, with little or no expression in epithelial cells.ConclusionsThese findings further substantiate the hypothesis that the cell of origin of EOC is tubal epithelium and that the beta isoform of folate receptor is primarily restricted to macrophages. Further, macrophages expressing FOLR2 may represent tumor associated or infiltrating macrophages (TAMs) in epithelial cancers.

Folate receptor-α (FOLR1) expression and function in triple negative tumors.

Folate receptor alpha (FOLR1) has been identified as a potential prognostic and therapeutic target in a number of cancers. A correlation has been shown between intense overexpression of FOLR1 in breast tumors and poor prognosis, yet there is limited examination of the distribution of FOLR1 across clinically relevant breast cancer subtypes. To explore this further, we used RNA-seq data from multiple patient cohorts to analyze the distribution of FOLR1 mRNA across breast cancer subtypes comprised of estrogen receptor positive (ER+), human epidermal growth factor receptor positive (HER2+), and triple negative (TNBC) tumors. FOLR1 expression varied within breast tumor subtypes; triple negative/basal tumors were significantly associated with increased expression of FOLR1 mRNA, compared to ER+ and HER2+ tumors. However, subsets of high level FOLR1 expressing tumors were observed in all clinical subtypes. These observations were supported by immunohistochemical analysis of tissue microarrays, with the largest number of 3+ positive tumors and highest H-scores of any subtype represented by triple negatives, and lowest by ER+ tumors. FOLR1 expression did not correlate to common clinicopathological parameters such as tumor stage and nodal status. To delineate the importance of FOLR1 overexpression in triple negative cancers, RNA-interference was used to deplete FOLR1 in overexpressing triple negative cell breast lines. Loss of FOLR1 resulted in growth inhibition, whereas FOLR1 overexpression promoted folate uptake and growth advantage in low folate conditions. Taken together, our data suggests patients with triple negative cancers expressing high FOLR1 expression represent an important population of patients that may benefit from targeted anti-FOLR1 therapy. This may prove particularly helpful for a large number of patients who would typically be classified as triple negative and who to this point have been left without any targeted treatment options.

Selectively Targeting Mutated NOTCH1 with a Folate-Thapsigargin Derivative

Gain-of-function mutations in NOTCH1, encoding a trans-membrane receptor involved in a transcriptional activation complex, are the most common genetic abnormality in human T-cell acute lymphoblastic leukemia (T-ALL). Thus, targeting NOTCH1 represents one promising therapeutic opportunity in T-ALL. The implementation of NOTCH1 pathway antagonists, however, poses several challenges secondary to concerns about inhibiting normal Notch receptors, leading to gut toxicity and secondary malignancies.We recently reported the results of a gene-expression based, NOTCH1 chemical genomic loss-of-function screen and a cell-based screen for cDNA enhancers of a NOTCH1 allele bearing a leukemia-associated mutation. SERCA (Sarco-endoplasmic reticulum Ca2+ ATPases) emerged at the intersection of these complementary approaches. We discovered that SERCA inhibition impairs the maturation of mutated NOTCH1 receptors and induces a G0/G1 arrest in NOTCH1-mutated human T-ALL cells. A small-molecule SERCA inhibitor, thapsigargin, a sesquiterpene-g-lactone, had on-target activity in mouse models of human T-ALL and also interfered with Notch signaling in Drosophila. Remarkably, thapsigargin preferentially inhibited mutated NOTCH1 receptors. This selectivity provides a therapeutic window not observed with other Notch pathway inhibitors, such as gamma-secretase inhibitors, that are equipotent inhibitors of mutated and wild-type receptors.While thapsigargin is a highly potent tool compound, in its native form, it is unlikely to have direct clinical application because of its global effects on intracellular Ca2+. Aberrant expression of folate receptors (FR) on the surface of cancer cells, including leukemia, has previously been reported, and efforts have been undertaken to leverage folate receptor-mediated endocytosis to deliver cancer therapeutics in vivo. We therefore developed a thapsigargin derivative conjugated with folic acid (JQ-FT) for preclinical testing in T-ALL.The FR gene family includes four members: FOLR1-4. The high affinity of FOLR1 and FOLR2 for folate binding, their endocytic capacity and their restricted expression pattern have prompted evaluation of folate-drug conjugates as a therapeutic approach in cancer. Analysis of gene-expression profiling suggested that FOLR2 is more highly and consistently expressed compared to FOLR1 in human T-ALL cell lines. We confirmed this finding by qRT-PCR across a panel of T-ALL cell lines and three primary patient samples. Surface expression of FOLR1 and FOLR2 measured by flow cytometry paralleled the transcriptional findings. We next validated the dependency of the uptake of a folate-FITC probe in T-ALL cells on active endocytosis via FR. First, we demonstrated that treatment of T-ALL cells with increasing concentrations of folate-FITC corresponds to an increase in measurable fluorescent signal by flow cytometry. The uptake is specific for FR as overexpression of FOLR1 or FOLR2 leads to increased, and folic acid competition decreased, fluorescent signal. Moreover, active endocytosis is supported by data demonstrating that folate-FITC fluorescence is not eliminated with an acidic wash step and that cold temperature does diminish internalization of the chemical probe.Next we demonstrated that increasing concentrations of JQ-FT impede the growth of T-ALL cells and induce apoptosis as measured by the activation of caspase 2. Moreover, the overexpression of ICN1, the intracellular active form of NOTCH1, partially rescues the small-molecule anti-proliferative effect indicating that the JQ-FT retains the on target activity similar to the underivatized thapsigargin. As observed with thapsigargin treatment of T-ALL cells, JQ-FT treatment decreased the levels of the intracellular active form of NOTCH1, ICN1, and the furin-processed transmembrane form of NOTCH1, while the full length precursor accumulated.In summary, these pre-clinical studies confirm the selective uptake of a folate-conjugated thapsigargin by an FR-mediated mechanism with the suppression of mutated NOTCH1 maturation and support the ongoing testing of this approach in mouse models of NOTCH1-mutated T-ALL. If successful, this approach would enhance the therapeutic window of thapsigargin as a NOTCH1 inhibitor providing dual selectivity: leukemia over normal cell and NOTCH1 mutated over wild-type receptors. DisclosuresNo relevant conflicts of interest to declare.

Abstract A27: The ovarian tumor tissue analysis (OTTA) consortium

Abstract Our understanding of ovarian cancer is complicated by disease heterogeneity. There are five major histological types, each with alterations in different pathways and distinct clinical outcomes. Even within these histological types there is clinical and molecular heterogeneity. The Ovarian Tumor Tissue Analysis (OTTA) consortium was established to validate prognostic markers for ovarian cancer in a large population of patients to allow stratification by histological type and molecular subtype. OTTA is currently a collaboration among 38 studies and has tissue microarrays (TMAs) from over 8,000 tumors available for immunohistochemical (IHC) analysis. In addition formalin fixed paraffin embedded (FFPE) tissue on slides or cores are available from the majority of OTTA studies for DNA and RNA analysis. Approximatley 60% of the cases are part of the Ovarian Cancer Association Consortium (OCAC) and have genotype data on over 200,000 single nucleotide polymorphisms and extensive epidemiological data. We recently performed IHC analysis of 12 markers using centralised staining and scoring to reduce heterogeneity and improve power to validate biomarkers. We showed that estrogen receptor and progesterone receptor were prognostic markers for endometrioid and high-grade serous carcinomas by performing the first robust subtype-specific analyses of these biomarkers in nearly 3000 patients. In contrast, FOLR1 expression was not associated with survival in 1507 high-grade serous carcinomas. We have shown that FFPE material from OTTA is suitable for RNA expression analysis on high through-put platforms such as the NanoString GX system. Expression analysis of 50 candidate genes in 244 high grade serous ovarian cancer cases has validated prognostic markers identified by The Cancer Genome Atlas (TCGA) project and investigated a series of novel candidate genes. IHC and RNA expression profiling from formalin fixed paraffin embedded (FFPE) tumor tissue is being used to better subclassify the ovarian tumors for further analysis. We would welcome groups with collections of ovarian tumor samples and corrsponding clinical data to join OTTA and participate in ongoing collaborative projects. Citation Format: Susan J. Ramus, Martin Köbel, Weiva Sieh, Michael S. Anglesio, Joshua Millstein, David D. Bowtell, James D. Brenton, Estrid Høgdall, Paul DP Pharoah, Ellen L. Goode, David G. Huntsman. The ovarian tumor tissue analysis (OTTA) consortium. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A27.

Gene Expression Analyses Support Fallopian Tube Epithelium as the Cell of Origin of Epithelial Ovarian Cancer

Folate receptor alpha (FOLR1/FRA) is reported to be overexpressed in epithelial ovarian cancers (EOC), especially the serous histotype. Further, while dysregulation of the folate-dependent 1-carbon cycle has been implicated in tumorogenesis, little is known relative to the potential mechanism of action of FOLR1 expression in these processes. We therefore investigated the expression of FOLR1, other folate receptors, and genes within the 1-carbon cycle in samples of EOC, normal ovary and fallopian tube on a custom TaqMan Low Density Array. Also included on this array were known markers of EOC such as MSLN, MUC16 and HE4. While few differences were observed in the expression profiles of genes in the 1-carbon cycle, genes previously considered to be overexpressed in EOC (e.g., FOLR1, MSLN, MUC16 and HE4) showed significantly increased expression when comparing EOC to normal ovary. However, when the comparator was changed to normal fallopian tube, these differences were abolished, supporting the hypothesis that EOC derives from fallopian fimbriae and, further, that markers previously considered to be upregulated or overexpressed in EOC are most likely not of ovarian origin, but fallopian in derivation. Our findings therefore support the hypothesis that the cell of origin of EOC is tubal epithelium.

Original article Expression of genes FOLR1, BAG1 and LAPTM4B in functioning and non-functioning pituitary adenomas

The mechanism of pathogenesis of adenomas pituitary is still unknown; differences between pituitary cells of different origin are observed. Identification of genes specific to pituitary adenomas should give better understanding of differences in their response to therapy, especially to radiotherapy. The aim of our study was to independently validate differences in the expression of FOLR1, BAG1, LAPTM4B between functioning (FA) and non-functioning (NFA) pituitary adenomas reported by microarray-based studies. Analysis of gene expression was performed by real-time quantitative PCR (QPCR) in 76 pituitary adenomas, 25 functioning and 51 non-functioning ones. The expression of the examined genes was normalized to the reference index, obtained by calculation of the geometric mean of reference genes expression: GUS-B, B2M, ACTB, EIF3S10, UBE2D2 and ATP6V1E. Two genes showed significant differences in expression between non-functioning adenomas and functioning ones (FA) (FOLR1 32.4 x greater p = 0.022, BAG1 2.2 x lower p = 0.0002). The expression of LAPTM4B (1.1 x lower) was only insignificantly changed. The expression of FOLR1 in all tumours (functioning and non-functioning) was higher in older patients (over 50 years of age) (p = 0.018). Expression of BAG1 was significantly lower in older patients (p = 0.015). In a subgroup of pure non-functioning adenomas there was a higher expression of FOLR1 in older patients (p = 0.006). Analysis of expression profiles and invasiveness of tumours did not reveal any significant differences both in non-functioning and functioning tumours. Among pituitary adenomas, the highest level of expression FOLR1 is seen in NFA which are negative by immunohistochemistry to all pituitary hormones while GH-producing adenomas are the only class of pituitary tumours where FOLR1 expression is virtually absent. For BAG1 we confirm a significantly higher expression in functioning (both PRL and GH producing) adenomas than non-functioning ones, while LAPTM4B does not exhibit any expression changes between different classes of pituitary tumours.

Inhibition of α Folate Receptor Resulting in a Reversal of Taxol Resistance in Nasopharyngeal Carcinoma

To further determine the role of FOLR1 in taxol resistance of nasopharyngeal carcinoma (NPC) and whether inhibition of FOLR1 expression reverses the taxol-resistant phenotype. Correlation study between gene expression and cancer cell survival. University hospital. Three taxol-resistant sub-cell lines with a different resistant index were established from the parental CNE-1 NPC cell line. The correlation between FOLR1 expression and taxol sensitivity was statistically analyzed. Inhibition of FOLR1 expression was carried out by RNA interference and by a FOLR1-specific monoclonal antibody, and taxol sensitivity was examined by colony formation assays. FOLR1 expression was also examined in 72 NPC patient specimens by immunohistochemistry. The levels of FOLR1 expression were positively and significantly correlated with a taxol resistance phenotype (P < .05). Inhibition of FOLR1 expression resulted in a significantly increased sensitivity of taxol to taxol-resistant NPC cells (P < .05). An increase of FOLR1 expression by gene transfection caused a taxol-resistant phenotype in parental NPC cells. The level of FOLR1 expression was found to be related to clinical stage in NPC tissue samples. These results suggest that FOLR1 plays an important role in taxol resistance of NPC cells.

Histone Crosstalk Directed by H2B Ubiquitination Is Required for Chromatin Boundary Integrity

Genomic maps of chromatin modifications have provided evidence for the partitioning of genomes into domains of distinct chromatin states, which assist coordinated gene regulation. The maintenance of chromatin domain integrity can require the setting of boundaries. The HS4 insulator element marks the 3′ boundary of a heterochromatin region located upstream of the chicken β-globin gene cluster. Here we show that HS4 recruits the E3 ligase RNF20/BRE1A to mediate H2B mono-ubiquitination (H2Bub1) at this insulator. Knockdown experiments show that RNF20 is required for H2Bub1 and processive H3K4 methylation. Depletion of RNF20 results in a collapse of the active histone modification signature at the HS4 chromatin boundary, where H2Bub1, H3K4 methylation, and hyperacetylation of H3, H4, and H2A.Z are rapidly lost. A remarkably similar set of events occurs at the HSA/HSB regulatory elements of the FOLR1 gene, which mark the 5′ boundary of the same heterochromatin region. We find that persistent H2Bub1 at the HSA/HSB and HS4 elements is required for chromatin boundary integrity. The loss of boundary function leads to the sequential spreading of H3K9me2, H3K9me3, and H4K20me3 over the entire 50 kb FOLR1 and β-globin region and silencing of FOLR1 expression. These findings show that the HSA/HSB and HS4 boundary elements direct a cascade of active histone modifications that defend the FOLR1 and β-globin gene loci from the pervasive encroachment of an adjacent heterochromatin domain. We propose that many gene loci employ H2Bub1-dependent boundaries to prevent heterochromatin spreading.

High‐affinity folate receptor in cardiac neural crest migration: A gene knockdown model using siRNA

Folate supplementation reduces the incidence of congenital heart defects, but the nature of this protective mechanism remains unclear. Immunolabeling demonstrated that the neural tube and neural crest (NC) cells were rich in the high-affinity folate receptor FOLR1and during the early stages of development FOLR1 was found principally in these cells. Suppression of Folr1 expression in the nascent cardiac NC by site-directed short-interfering RNA (siRNA) altered cardiac NC cell mitosis and subsequent migration patterns leading to abnormal development of the pharyngeal arch arteries (PAA) and outflow tract. qPCR analysis demonstrated that the siRNA treatment significantly reduced Folr1 24 hr after treatment. These treatments also significantly reduced mitosis in the neural tube, but adjacent, nontreated areas were unaffected. In summary, a brief reduction in the expression of Folr1 during a critical stage of NC development had long-term consequences for the development of the PAA and outflow tract.

108 CEC and CTC in stage IV breast cancers treated with bevacizumab (Bv) and chemotherapy (CT)

Folate receptor alpha (FOLR1) has been identified as a potential prognostic and therapeutic target in breast cancer. The limited studies evaluating the role of FOLR1 in breast cancer have shown that FOLR1 protein expression is enriched in triple-negative breast cancer (TNBC) and associated with poor prognosis in all breast cancer types. Newly developed anti-FOLR1 therapy could potentially be used in patients with TNBC for whom few therapeutic options exist. We sought to evaluate FOLR1 protein expression in a cohort of patients with TNBC to determine its prevalence and prognostic value.Immunohistochemistry was performed for FOLR1 in 76 cases of primary TNBC. Membranous staining in ≥ 5% of cells was deemed positive in a given case. Statistical analyses correlating FOLR1 protein expression with clinicopathologic parameters and clinical outcome (disease-free survival and overall survival) were performed.A total of 76 cases of primary TNBC were studied. Most cases were negative for FOLR1 (80.3%; 61/76). FOLR1 expression did not correlate with any clinicopathologic parameters. FOLR1 expression was significantly correlated with decreased disease-free survival (hazard ratio, 2.61; 95% confidence interval, 0.96-7.09; P = .0497 log-rank test). Although FOLR1 expression trended towards decreased overall survival, it was not statistically significant (hazard ratio, 1.99; 95% confidence interval, 0.62-6.36; P > .05 log-rank test).We found a lower incidence of FOLR1 expression in TNBC compared with other studies; however, these patients may benefit from anti-folate therapy as other targeted therapies are not available. Although no correlation between FOLR1 expression and standard clinicopathologic parameters was identified, our findings suggest that FOLR1 expression is prognostically significant in TNBC.

Different histological types of non‐small cell lung cancer have distinct folate and DNA methylation levels

Aberrant DNA methylation is a commonly observed epigenetic change in lung cancer. Folate has been suggested to play a role in the homeostasis of DNA methylation and has also been implicated in cancer chemotherapy. We investigated a possible role for folate in DNA methylation by measuring folate concentrations in tumors and adjacent normal tissues from 72 non-small cell lung cancer (NSCLC) patients. These were compared to DNA methylation levels and to clinicopathological features. Folate concentrations were determined as the sum of 5,10-methylenetetrahydrofolate and tetrahydrofolate. The MethyLight assay was used to quantitate methylation in promoter regions of P16(CDKN2A), APC, CDH13, RARB, RASSF1, RUNX3, and MYOD1. Methylation of LINE-1 repeats was used as a surrogate for global methylation. Folate levels in tumors correlated positively with LINE-1, CDH13, and RUNX3 methylation. Folate concentrations and methylation of LINE-1, RASSF1, and RUNX3 were significantly higher in adenocarcinoma compared to squamous cell carcinoma (SCC). Two sets of array-based data retrieved from the Gene Expression Omnibus consistently showed that expression of FOLR1, a folate transport enzyme, and GGH, an enzyme that prevents folate retention, were higher and lower, respectively, in adenocarcinomas compared to SCC. This was independently validated by quantitative RT-PCR in 26 adenocarcinomas and 13 SCC. Our results suggest that folate metabolism plays a role in aberrant DNA methylation in NSCLC. The histological subtype differences in folate concentration and DNA methylation observed here were associated with distinct expression patterns for folate metabolizing enzymes. These findings may have clinical applications for histology-directed chemotherapy with fluoropyrimidine and anti-folates in NSCLC.

Expression of the folate receptor genes FOLR1 and FOLR3 differentiates ovarian carcinoma from breast carcinoma and malignant mesothelioma in serous effusions

The objective of this study was to analyze the diagnostic and clinical role of the folate receptor-alpha (FOLR1) and folate receptor-gamma (FOLR3) genes in effusion cytology. Expression of the FOLR1 protein product, FR-alpha, was additionally studied. Ninety-one effusions (71 ovarian carcinomas, 10 breast carcinomas, 10 malignant mesotheliomas) were assayed for FOLR1 and FOLR3 gene expression using quantitative polymerase chain reaction. FR-alpha expression was analyzed using flow cytometry. Ovarian carcinoma expression levels were analyzed for association with clinicopathologic parameters and survival. Quantitative polymerase chain reaction analysis showed significantly higher FOLR1and FOLR3 mRNA levels in ovarian carcinomas compared with both breast carcinomas and mesotheliomas (P < .001). FOLR1 and FOLR3 mRNA levels were directly interrelated in ovarian carcinoma (P < .001). FR-alpha protein levels were similarly higher in ovarian carcinoma compared with the 2 other cancer types (P < .001). FOLR1and FOLR3 mRNA and FR-alpha protein expression in ovarian carcinoma effusions showed no association with clinical parameters or survival. Our data suggest that folate receptor levels effectively differentiate ovarian carcinoma from other cancers affecting the serosal cavities and that folate receptor genes are coexpressed in this tumor. The high expression of folate receptors in ovarian carcinoma supports their validity as molecular therapeutic targets in this disease.

Regulation of folate receptor 1 gene expression in the visceral endoderm

Nutrient supply to the developing mammalian embryo is a fundamental requirement. Before completion of the chorioallantoic placenta, the visceral endoderm plays a crucial role in nurturing the embryo. We have found that visceral endoderm cells express folate receptor 1, a high-affinity receptor for the essential micronutrient folic acid, suggesting that the visceral endoderm has an important function for folate transport to the embryo. The mechanisms that direct expression of FOLR1 in the visceral endoderm are unknown. Sequences were tested for transcriptional activation capabilities in the visceral endoderm utilizing reporter gene assays in a cell model for extraembryonic endoderm in vitro, and in transgenic mice in vivo. With F9 embryo carcinoma cells as a model for extraembryonic endoderm, we demonstrate that the P4 promoter of the human FOLR1 gene is active during differentiation of the cells towards visceral endoderm. However, transgenic mouse experiments show that promoter sequences alone are insufficient to elicit reporter gene transcription in vivo. Using sequence conservation as guide to choose genomic sequences from the human FOLR1 gene locus, we demonstrate that the sequence termed F1CE2 exhibits specific enhancer activity in F9 cells in vitro, in the visceral endoderm, and later the yolk sac in transgenic mouse embryos in vivo. We further show that the transcription factor HNF4-alpha can activate this enhancer sequence. We have identified a transcriptional enhancer sequence from the FOLR1 locus with specific activity in vitro and in vivo, and suggest that FOLR1 is a target for regulation by HNF4-alpha.

Expression Status of Folate Receptor α Is Significantly Correlated with Prognosis in Non-Small-Cell Lung Cancers

To evaluate the prognostic value of folate receptor alpha (FOLR1) and/or reduced folate carrier (RFC1) expression, which are well-characterized folate transporters, in completely resected non-small-cell lung cancer (NSCLC). We quantitatively examined gene expression of FOLR1 and RFC1 in surgical specimens resected from NSCLC patients. A total of 119 consecutive patients from January 2003 to June 2004 were included. In adenocarcinoma, the FOLR1 gene expression was downregulated in smokers and male patients (P = 0.006 and P < 0.001, respectively). In addition, FOLR1 expression values in patients with well-differentiated, early p-stage, pT1, pN0, EGFR mutant, and p53 wild-type cancers were significantly higher than those for poorly differentiated, advanced p-stage, pT2-4, pN1-3, EGFR wild-type, and p53 mutant (P < 0.001, P < 0.001, P < 0.001, P = 0.002, P < 0.001 and P = 0.023, respectively). In squamous cell carcinoma, FOLR1 expression values in patients with pN1-3 was significantly higher than those with pN0 (P = 0.037). Moreover, the 3-year survival rate and disease-free survival rate of high-FOLR1-expressing patients (94.7% and 75.4%) were significantly higher than those of low-FOLR1-expressing patients (80.9% and 60.8%) (P = 0.008 and P = 0.038). A multivariate analysis confirmed that high FOLR1 expression was an independent and significant factor predicting a favorable prognosis (P = 0.043). Higher levels of FOLR1 appear to be associated with better prognoses for patients with lung adenocarcinomas.

Overexpression of folate binding protein and mesothelin are associated with uterine serous carcinoma

Purpose Folate receptor alpha (FOLR1) is a membrane bound receptor involved in the transport of folate as well as other regulatory cellular processes. The purpose of this study was to examine the expression of FOLR1 in uterine cancers and to identify changes in gene expression that are associated with overexpression of FOLR1. Experimental design Fifty-eight frozen uterine cancer specimens were stained for FOLR1 using immunohistochemistry and results were correlated with transcript expression noted on quantitative PCR. Total RNA from 16 cases of uterine serous carcinoma (USC) was analyzed for gene expression using the Affymetrix HG-U133A and HG-U133B GeneChip set. USCs overexpressing FOLR1 were compared to cancers with an absence of FOLR1 using binary comparison and template matching of data was used to identify genes that correlate with FOLR1 expression. Selected targets from this analysis were evaluated by quantitative PCR as well as in an independent set of USC represented in quadruplicate on a tissue microarray (TMA). Results Overexpression of FOLR1 was observed in 11/16 (69%) of USC and 0/10 normal endometrium cases using frozen tissue specimens. Binary comparison between FOLR1 positive and negative cases identified 121 genes altered by 2-fold at p < 0.01 of which 45 are well correlated with FOLR1 expression pattern. Using quantitative PCR, both mesothelin (MSLN) and PTGS1 (COX1) were significantly increased in FOLR1 overexpressing tumors ( p = 0.014 and p = 0.006 respectively). TMA confirmed that overexpression of FOLR1 and MSLN respectively occurred in 23/48 (48%) and 17/54 (32%) of pure USC. Conclusion Both FOLR1 and MSLN are cell surface targets that are co-expressed at high levels in USC and are appealing targets for biologic therapy.

The role of folate receptor α in cancer development, progression and treatment: Cause, consequence or innocent bystander?

Folate receptor alpha (FRalpha) is a membrane-bound protein with high affinity for binding and transporting physiologic levels of folate into cells. Folate is a basic component of cell metabolism and DNA synthesis and repair, and rapidly dividing cancer cells have an increased requirement for folate to maintain DNA synthesis, an observation supported by the widespread use of antifolates in cancer chemotherapy. FRalpha levels are high in specific malignant tumors of epithelial origin compared to normal cells, and are positively associated with tumor stage and grade, raising questions of its role in tumor etiology and progression. It has been suggested that FRalpha might confer a growth advantage to the tumor by modulating folate uptake from serum or by generating regulatory signals. Indeed, cell culture studies show that expression of the FRalpha gene, FOLR1, is regulated by extracellular folate depletion, increased homocysteine accumulation, steroid hormone concentrations, interaction with specific transcription factors and cytosolic proteins, and possibly genetic mutations. Whether FRalpha in tumors decreases in vivo among individuals who are folate sufficient, or whether the tumor's machinery sustains FRalpha levels to meet the increased folate demands of the tumor, has not been studied. Consequently, the significance of carrying a FRalpha-positive tumor in the era of folic acid fortification and widespread vitamin supplement use in countries such as Canada and the United States is unknown. Epidemiologic and clinical studies using human tumor specimens are lacking and increasingly needed to understand the role of environmental and genetic influences on FOLR1 expression in tumor etiology and progression. This review summarizes the literature on the complex nature of FOLR1 gene regulation and expression, and suggests future research directions.