Normal Somatic Cells Research Articles

The telomerase inhibitor MST-312 synergistically enhances the apoptotic effect of doxorubicin in pre-B acute lymphoblastic leukemia cells

BackgroundThere have been consistent efforts in concomitant administration of chemotherapeutic agents such as doxorubicin beside other more tumor-specific drugs in order to increase sensitivity of tumor cells to the cytotoxicity of doxorubicin and reduce necessary chemotherapeutic dosage. Telomerase-targeted therapy for cancer has received great attention because telomerase is expressed in almost all cancer cells but is inactive in most normal somatic cells. The present study was aimed to investigate the effect of telomerase inhibitor MST-312, a chemically modified derivative of epigallocatechin gallate (EGCG), on doxorubicin-induced apoptosis in pre-B acute lymphoblastic leukemia cells. Materials and methodsThe pre-B ALL cell lines (NALM-6 and REH) were cultured and treated with MST-312 and doxorubicin, separately and in combination. Cell viability was measured by trypan blue staining and MTT assay. Annexin-V/7-AAD staining by flow cytometry was used for evaluation of apoptosis. Gene expression of hTERT, c-Myc, Bax and Bcl-2, was detected by the quantitative Real-Time PCR. ResultsOur results showed that MST-312 exerted dose-dependent short-term cytotoxic and apoptotic effects on pre-B ALL cells. Combination of MST-312 and doxorubicin synergistically enhanced the cytotoxicity and apoptosis of both NALM-6 and REH cells. Consistently, apoptosis induction by the combined treatment was associated with significant reduction of Bcl-2, c-Myc and hTERT, and a marked increase of Bax mRNA expression levels, compared with the single treatments. ConclusionsOur data suggest that the synergistic anticancer effect induced by combination of MST-312 and doxorubicin represents a novel treatment strategy for pre-B ALL.

Abstract 440: Single-cell whole-genome sequencing reveals somatic mutation signatures in normal somatic cells predictive of cancer later in life

Abstract Single-cell sequencing for analyzing DNA mutations across the genome in somatic tissues is critically important for studying development, cancer and aging. However, current procedures are prone to artifacts and to date a reliable protocol for single-cell somatic mutation analysis remains to be developed. We address the two largest sources of artifacts, i.e., DNA denaturation-related cytosine deamination and allelic bias-driven whole genome amplification errors. We first reconfigured multiple displacement amplification (MDA) into an efficient protocol for whole genome amplification of single cells without cytosine deamination artifacts, i.e., Single Cell MDA (SCMDA). We then developed a new single-cell SNV caller (SCcaller) that distinguishes real somatic mutations and amplification errors by utilizing a SNP-based localized estimate of allelic amplification bias. The procedure was validated by comparing SCMDA-amplified single cells with unamplified clones derived from single cells from the same population. Using this highly accurate single-cell whole-genome sequencing method we analyzed human B lymphocytes from donors varying in age from birth to over 100 year, studying both genome distribution and functional impact of base substitution mutations. Mutations per cell were found to increase with age from less than 500 in cord blood to over 3,000 in cells from individuals over 100. While overall mutations were randomly distributed across the genome with all chromosomes equally affected, 24 hotspot regions were identified, five of which were part of immunoglobulin variable regions subject to somatic hypermutation. Age-related mutation accumulation was found to be significantly slower in genomic sequences directly involved in cellular function, such as exons and gene regulatory sequences. Still, on average, B lymphocytes from aged individuals contained 3-15 damaging mutations in the transcribed part of the exome identified by RNA-seq, plus 15-50 mutations in transcription factor binding sites identified by ATAC-seq. Analysis of the spontaneous mutation spectra in these normal cells revealed signatures similar to those previously found associated with B cell leukemia and other cancers. These results indicate that cancer risk is already part of age-related mutation accumulation in normal cells. Taken together, our single-cell sequencing method provides a firm foundation for analyzing cellular genetic heterogeneity in normal human tissues. Citation Format: Xiao Dong, Lei Zhang, Moonsook Lee, Alexander Y. Maslov, Jan Vijg. Single-cell whole-genome sequencing reveals somatic mutation signatures in normal somatic cells predictive of cancer later in life [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 440.

Abstract 470: p53 isoform delta133p53 in tumor senescence

Abstract The tumor suppressor gene TP53 expresses at least 12 isoforms due to alternative splicing, alternative initiation of translation, and alternative promoter usage. These isoforms include p53β and Δ133p53, which can modulate p53 transcriptional activity and apoptosis. The p53 isoform Δ133p53 is expressed in normal tissue, but is abnormally expressed in several cancer types. However, its role in tumor progression is still unclear. Normal somatic cells undergo a limited number of cell divisions, eventually leading to an irreversible proliferative growth arrest known as replicative senescence. Replicative senescence can act as a tumor suppressor mechanism, thus constituting a critical barrier to tumor progression in vivo. Previous studies showed that, p53β and Δ133p53 are endogenous regulators of replicative senescence in normal human fibroblasts. Interestingly, expression of Δ133p53 is upregulated in several cancer types. For instance, enhanced levels of Δ133p53 are associated with inhibition of cellular senescence in colon carcinoma as well as with progression of premalignant colon adenomas to colon carcinomas. However, little is known about the role of Δ133p53 in cancer. We are studying the roles of Δ133p53 in proliferation and senescence of cancer cell lines. We hypothesized that depletion of otherwise enhanced Δ133p53 isoform induces cellular growth arrest and increases the secretion of senescence-associated secretory phenotype (SASP) pro-inflammatory cytokines such as IL-6 and IL-8 of cancer cell lines, and thus Δ133p53 may be a therapeutic target to repress tumor cell growth. Citation Format: Natalia Von Muhlinen, Jessica Beck, Curtis C. Harris. p53 isoform delta133p53 in tumor senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 470.

Abstract 4392: Novel combination of Di-indolylmethane (DIM) and dexamethasone induces apoptosis in myeloma cells by modulating the telomerase activity

Abstract Background and Objectives Multiple myeloma (MM) is a B-cell malignancy involving excessive proliferation and accumulation of atypical plasma cells specifically in the bone marrow (BM) niche. Multiple primary and secondary genetic alterations play crucial role in deregulating the biology of myeloma cells giving them a malignant phenotype, hence generating a need for novel efficacious and safe therapeutic agents. Increased expression of telomerase and higher telomerase activity (RTA) has been found in majority of cancer but displays very low activity in majority of normal somatic cells. Therefore, this maiden attempt unveils the therapeutic potential of Di-indolylmethane alone or in combination with dexamethasone by targeting telomerase activity in myeloma cells. Methodology In this study, two myeloma cell lines (RPMI8226 & U266) were procured from ATCC (Manassas, VA). Di-indolylmethane (DIM) and dexamethasone (Dexa) were purchased from Sigma Aldrich (St. Louis, MO). In in-vitro study, IC50 concentration of DIM and Dexa on both cells was determined using MTT dye. Cell-cycle and apoptosis assay was performed to determine the anti-proliferative and apoptotic effects of DIM and Dexa alone or in combination. Further, caspase-3/7 activity and mitochondrial membrane potential (MMP) was determined to assessed pathway of apoptosis. Effects of single drug or combination on telomerase activity were also assessed in both myeloma cells. Results Results demonstrated that, DIM showed an IC50 conc. of 40μM in both myeloma cells while these cells were resistant to Dexa even at higher dose. In cell cycle assay, significant increase in sub-G0/G1 phase were observed with increasing dose of DIM while combination of DIM and Dexa significantly increased sub-G0/G1 cell populations in comparison to vehicle control. Similarly annexin-V/PI apoptosis assay also demonstrated increased apoptosis of both myeloma cells. Further, DIM alone or in combination with Dexa demonstrated drastic increased caspase-3/7 activity and significant decrease in MMP in both myeloma cells. Additionally, increasing dose of DIM significantly decreased telomerase activity and combination of DIM and Dexa was much more potent in decreasing telomerase activity in comparison to vehicle treatment and DIM alone. Conclusions In nutshell, this maiden attempt provided initial evidences of therapeutic potential of DIM as novel anticancer agent alone or in combination with standard chemotherapeutic agent Dexa. Further, novel combination of DIM and Dexa was much more potential in inducing apoptosis cell death and chemo-sensitizing myeloma cells than single drug treatment regime. Also intrinsic pathway of apoptosis was followed by DIM in inducing apoptosis in myeloma. Furthermore, decreased TA by DIM emphasized its potential as novel molecular therapeutic target which need further validation. Citation Format: Raman Kumar, Nidhi Gupta, . Himani, Alpana Sharma. Novel combination of Di-indolylmethane (DIM) and dexamethasone induces apoptosis in myeloma cells by modulating the telomerase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4392.

Homologous recombination occurs frequently at innate GT microsatellites in normal somatic and germ cells in vivo

BackgroundIn somatic cells, homologous recombination (HR) is a rare event caused by eventual DNA double-strand breaks (DSBs). In contrast, germ cells show high frequency of HR caused by programmed DSBs. Microsatellites are prone to DSBs during genome replication and, thereby, capable of promoting HR. It remains unclear whether HR occurs frequently at microsatellites both in normal somatic cells and germ cells in a similar manner.ResultsBy examining the linkage pattern of multiple paternal and maternal markers flanking innate GT microsatellites, we measured HR at the GT microsatellites in various somatic cells and germ cells in a goldfish intraspecific heterozygote. During embryogenesis, the HR products accumulate gradually with the increase of the number of cell divisions. The frequency of HR at the GT microsatellites in advanced embryos, adult tissues and germ cells is surprisingly high. The type of exchanges between the homologous chromosomes is similar in normal advanced embryos and germ cells. Furthermore, a long GT microsatellite is more active than a short one in promoting HR in both somatic and germ cells.ConclusionsHR occurs frequently at innate GT microsatellites in normal somatic cells and germ cells in a similar manner.

Ratiometric Fluorescent Biosensor for Visual Discrimination of Cancer Cells with Different Telomerase Expression Levels.

Telomerase is inactive in normal somatic cells but highly activated in tumor cells to maintain their indefinite proliferation and immortal phenotype. As a specific marker for the generation and progress of almost all tumors, the detection of telomerase activity by classical PCR techniques has served in the biological research of tumors. However, the detection of in situ telomerase activity in cell extracts to evaluate the malignancy, progress, and metastasis of tumors remains a daunting challenge. Here, a precisely designed FRET-based ratiometric fluorescent oligonucleotide probe has achieved high-fidelity detection of telomerase activity for accurate discrimination of different cancer cells toward advanced diagnosis of tumors. Our method is superior to other methods in its capabilities to quantify telomerase activity in cell extracts and visualize various tumor cell extracts with different telomerase expression levels by the naked eye for clinical diagnosis. In particular, the ratiometric fluorescent probe used in the assay could exclude other experimental factors influence, and further avoid false positive signal generation. The method reported here could provide a reliable, accurate, and convenient way in medical diagnostics and therapeutic response assessment.

Overexpression of FOXQ1 enhances anti-senescence and migration effects of human umbilical cord mesenchymal stem cells in vitro and in vivo.

Mesenchymal stem cells (MSCs) are unique precursor cells characterized by active self-renewal and differentiation potential. These cells offer the advantages of ease of isolation and limited ethical issues as a resource and represent a promising cell therapy for neurodegenerative diseases. However, replicative senescence during cell culture as well as low efficiency of cell migration and differentiation after transplantation are major obstacles. In our previous study, we found that FOXQ1 binds directly to the SIRT1 promoter to regulate cellular senescence and also promotes cell proliferation and migration in many tumor cell lines. Currently, little is known about the effects of FOXQ1 on normal somatic cells. Therefore, we examine the effects of FOXQ1 on senescence and migration of MSCs. Lentiviral vector-mediated overexpression of FOXQ1 in human umbilical cord mesenchymal stem cells (hUC-MSCs) resulted in enhanced cell proliferation and viability. Furthermore, the expression of proteins and markers positively associated with senescence (p16, p21, p53) was reduced, whereas expression of proteins negatively associated with senescence (SIRT1, PCNA) was promoted. Following transplantation of hUC-MSCs overexpressing FOXQ1 in an animal model of Alzheimer's disease (APPV717I transgenic mice) resulted in amelioration of the effects of Alzheimer's disease (AD) on cognitive function and pathological senescence accompanied the increased numbers of hUC-MSCs in the AD brain. In conclusion, FOXQ1 overexpression promotes anti-senescence and migration of hUC-MSCs in vitro and in vivo. These findings also suggest that this strategy may contribute to optimization of the efficiency of stem cell therapy.

Mandatory role of HMGA1 in human airway epithelial normal differentiation and post-injury regeneration.

Due to high levels of expression in aggressive tumors, high mobility group AT-hook 1 (HMGA1) has recently attracted attention as a potential anti-tumor target. However, HMGA1 is also expressed in normal somatic progenitor cells, raising the question: how might systemic anti-HMGA1 therapies affect the structure and function of normal tissue differentiation? In the present study, RNA sequencing data demonstrated HMGA1 is highly expressed in human airway basal stem/progenitor cells (BC), but decreases with BC differentiation in air-liquid interface cultures (ALI). BC collected from nonsmokers, healthy smokers, and smokers with chronic obstructive pulmonary disease (COPD) displayed a range of HMGA1 expression levels. Low initial expression levels of HMGA1 in BC were associated with decreased ability to maintain a differentiated ALI epithelium. HMGA1 down-regulation in BC diminished BC proliferation, suppressed gene expression related to normal proliferation and differentiation, decreased airway epithelial resistance, suppressed junctional and cell polarity gene expression, and delayed wound closure of airway epithelium following injury. Furthermore, silencing of HMGA1 in airway BC in ALI increased the expression of genes associated with airway remodeling in COPD including squamous, epithelial-mesenchymal transition (EMT), and inflammatory genes. Together, the data suggests HMGA1 plays a central role in normal airway differentiation, and thus caution should be used to monitor airway epithelial structure and function in the context of systemic HMGA1-targeted therapies.

Non-coding RNAs in the reprogramming of glucose metabolism in cancer

Proliferating cancer cells reprogram their metabolic circuitry to thrive in an environment deficient in nutrients and oxygen. Cancer cells exhibit a higher rate of glucose metabolism than normal somatic cells, which is achieved by switching from oxidative phosphorylation to aerobic glycolysis to meet the energy and metabolites demands of tumour progression. This phenomenon, which is known as the Warburg effect, has generated renewed interest in the process of glucose metabolism reprogramming in cancer cells. Several regulatory pathways along with glycolytic enzymes are responsible for the emergence of glycolytic dependence. Non-coding (nc)RNAs are a class of functional RNA molecules that are not translated into proteins but regulate target gene expression. NcRNAs have been shown to be involved in various biological processes, including glucose metabolism. In this review, we describe the regulatory role of ncRNAs–specifically, microRNAs and long ncRNAs–in the glycolytic switch and propose that ncRNA-based therapeutics can be used to inhibit the process of glucose metabolism reprogramming in cancer cells.

AKAP4, SPAG9 and NY-ESO-1 in Iranian Colorectal Cancer Patients as Probable Diagnostic and Prognostic Biomarkers

Background and objectives:Colorectal cancer (CRC) is the most common gastrointestinal cancer and the second leading cause of cancer death in women in the world. Cancer-Testis Antigens (CTAs) are a group of tumor-associated proteins which typically are expressed in normal reproductive cells of men, but their expression in normal somatic cells is silenced. CTAs, due to their limited expression pattern, are considered as promising targets for cancer diagnosis and immuno-therapy.Methods:Expression of AKAP4, SPAG9 and CTAG1B genes from the CTAs family was studied in both tumor and normal tissues of 62 Iranian CRC patients by RT-PCR with the aim of finding biomarkers for early detection and anticipated progression. Statistical analysis was performed SPSS software V22.0 to assess the significance of any associations.Results:Elevated expression of SPAG9 and AKAP4 genes was observed in approximately 66% and 44% of tumours, respectively, as compared to adjacent non-cancerous tissues. While a significant association was found between AKAP4 gene expression and metastasis (P-value: 0.045), expression of the CTAG1B (NY-ESO-1) gene was not observed in our cases.Conclusion:AKAP4 and SPAG9 genes may find use as diagnostic biomarkers for CRC and AKAP4 may play an important role in progression to metastasis.

A Novel Long Non-Coding RNA in the hTERT Promoter Region Regulates hTERT Expression.

A novel antisense transcript was identified in the human telomerase reverse transcriptase (hTERT) promoter region, suggesting that the hTERT promoter is bidirectional. This transcript, named hTERT antisense promoter-associated (hTAPAS) RNA, is a 1.6 kb long non-coding RNA. hTAPAS transcription is initiated 167 nucleotides upstream of the hTERT transcription start site and is present in both the nucleus and the cytoplasm. Surprisingly, we observed that a large fraction of the hTERT polyadenylated RNA is localized in the nucleus, suggesting this might be an additional means of regulating the cellular abundance of hTERT protein. Both hTAPAS and hTERT are expressed in immortalized B-cells and human embryonic stem cells but are not detected in normal somatic cells. hTAPAS expression inversely correlates with hTERT expression in different types of cancer samples. Moreover, hTAPAS expression is not promoted by an hTERT promoter mutation (-124 C>T). Antisense-oligonucleotide mediated knockdown of hTAPAS results in an increase in hTERT expression. Conversely, ectopic overexpression of hTAPAS down regulates hTERT expression, suggesting a negative role in hTERT gene regulation. These observations provide insights into hTAPAS as a novel player that negatively regulates hTERT expression and may be involved in telomere length homeostasis.

ETS1, nucleolar and non-nucleolar TERT expression in nevus to melanoma progression.

TERT (telomerase reverse transcriptase) is the catalytic component of telomerase. TERT shows little expression in normal somatic cells but is commonly re-expressed in cancers, facilitating immortalization. Recently-discovered TERT promoter mutations create binding sites for ETS-family transcription factors to upregulate TERT. ETS1 is reported to be important for TERT upregulation in melanoma. However it is unclear when in melanoma progression TERT and ETS1 proteins are expressed. To elucidate this question, ETS1 and TERT immunohistochemistry were performed on a panel of benign (n=27) and dysplastic nevi (n=34), radial growth phase (n=29), vertical growth phase (n=25) and metastatic melanomas (n=27). Lesions were scored by percentage of positive cells. ETS1 was readily detectable in all lesions, but not in normal melanocytes. TERT was located in either the nucleolus, the nucleoplasm (non-nucleolar) or both. Non-nucleolar TERT increased in prevalence with progression, from 19% of benign nevi to 78% of metastases. It did not however correlate with cell proliferation (Ki-67 immunostaining), nor differ significantly in prevalence between primary melanomas with or without a TERT promoter mutation. These results demonstrate that ETS1 is expressed very early in melanoma progression, and interestingly only non-nucleolar TERT correlates clearly in prevalence with melanoma progression. It can be acquired at various stages and by mechanisms other than promoter mutations.

Survival of BRCA2-Deficient Cells Is Promoted by GIPC3, a Novel Genetic Interactor of BRCA2.

BRCA2 loss-of-heterozygosity (LOH) is frequently observed in BRCA2-mutated tumors, but its biallelic loss causes embryonic lethality in mice and inhibits proliferation of normal somatic cells. Therefore, it remains unclear how loss of BRCA2 contributes to tumorigenesis. One possibility is that mutation in potential genetic interactors of BRCA2, such as TRP53, is required for cell survival/proliferation in the absence of BRCA2. In this study, using an insertional mutagenesis screen in mouse embryonic stem cells (mESC), we have identified GIPC3 (GAIP-interacting protein C-terminus 3) as a BRCA2 genetic interactor that contributes to survival of Brca2-null mESC. GIPC3 does not compensate for BRCA2 loss in the repair of double-strand breaks. Mass-spectrometric analysis resulted in the identification of G-protein signaling transducers, APPL1 and APPL2, as potential GIPC3-binding proteins. A mutant GIPC3 (His155Ala) that does not bind to APPL1/2 failed to rescue the lethality of Brca2-null mESC, suggesting that the cell viability by GIPC3 is mediated via APPL1/2. Finally, the physiological significance of GIPC3 as a genetic interactor of BRCA2 is supported by the observation that Brca2-null embryos with Gipc3 overexpression are developmentally more advanced than their control littermates. Taken together, we have uncovered a novel role for GIPC3 as a BRCA2 genetic interactor.

Inhibition of survivin expression after using oxaliplatin and vinflunine to induce cytogenetic damage in vitro in lymphocytes from colon cancer patients and healthy individuals.

Chemotherapy drugs usually inflict a lethal dose to tumour cells with the consequence that these cells are being killed by cell death. However, each round of chemotherapy also causes damage to normal somatic cells. The DNA cross-linking agent oxaliplatin (OXP), which causes DNA double-strand breaks, and vinflunine (VFN), which disrupts the mitotic spindle, are two of these chemotherapy drugs which were evaluated in vitro using peripheral lymphocytes from colorectal cancer patients and healthy individuals to determine any differential response. Endpoints examined included micronucleus (MN) induction using the cytokinesis-blocked micronucleus (CBMN) assay and pancentromeric fluorescence in situ hybridisation. Also, survivin expression was monitored since it regulates the mitotic spindle checkpoint and inhibits apoptosis. OXP produced cytogenetic damage (micronuclei in binucleated cells) via its clastogenic but also previously unknown aneugenic action, possibly through interfering with topoisomerase II, whilst VFN produced micronuclei in mononucleated cells because of incomplete karyokinesis. Survivin expression was found to be significantly reduced in a concentration-dependent manner by not only OXP but surprisingly also VFN. This resulted in large numbers of multinucleated cells found with the CBMN assay. As survivin is upregulated in cancers, eliminating apoptosis inhibition might provide a more targeted chemotherapy approach; particularly, when considering VFN, which only affects cycling cells by inhibiting their mitotic spindle, and alongside possibly other pro-apoptotic compounds. Hence, these newly found properties of VFN -the inhibition of survivin expression-might demonstrate a promising chemotherapeutic approach as VFN induces less DNA damage in normal somatic cells compared to other chemotherapeutic compounds.

ID3010 Wnt antagonism in cancer stem cells- targeting stemness of cancers via the Wnt-BETA catenin pathway

Cancer stem cells (CSCs) are hypothesized to be the pathological counterpart of normal somatic tissue stem cells. The CSC model proposes that tumours are hierarchically organised with a subset of tumour cells at their apex, which possess self-renewal and multilineage differentiation potential. Cancer stem cells, unlike the bulk of the cells within the tumor, are elusive to drug treatment and are unaffected on chemo and radiotherapy. These self-renewing cells are responsible for the flare up of cancer and remission long after treatment. Cancer stem cells have a capacity for unlimited self-renewal, as well as the ability to initiate and drive tumor progression in an animal model.
 Activated Wnt/β-Catenin signaling is a key feature of epithelial cancers and is critical for metastasis and epithelial-mesenchymal transition (EMT) , a signature trait of CSCs. We explored the effect of the Wnt antagonist, secreted frizzled related protein 4 (sFRP4) in CSCs in gliomas. We found that sFRP4 chemo-sensitizes GSC-enriched cells to commonly used drugs, by the reversal of EMT and by decreasing drug effluxers. sFRP4 acts at multiple levels of the Wnt- β-Catenin and the Wnt- calcium pathways in inhibiting CSCs. We also identified a novel mechanism of action of sFRP4. These findings could be exploited for designing better targeted strategies to improve chemo-response and eventually eliminate glioblastoma CSCs.

Nanogap‐Rich Au Nanowire SERS Sensor for Ultrasensitive Telomerase Activity Detection: Application to Gastric and Breast Cancer Tissues Diagnosis

Telomerase has attracted much attention as a universal cancer biomarker because telomerase is overexpressed in more than 85% of human cancer cells while suppressed in normal somatic cells. Since a strong association exists between telomerase activity and human cancers, the development of effective telomerase activity assay is critically important. Here, a nanogap‐rich Au nanowire (NW) surface‐enhanced Raman scattering (SERS) sensor is reported for detection of telomerase activity in various cancer cells and tissues. The nanogap‐rich Au NWs are constructed by deposition of nanoparticles on single‐crystalline Au NWs and provided highly reproducible SERS spectra. The telomeric substrate (TS) primer‐attached nanogap‐rich Au NWs can detect telomerase activity through SERS measurement after the elongation of TS primers, folding into G‐quadruplex structures, and intercalation of methylene blue. This sensor enables us to detect telomerase activity from various cancer cell lines with a detection limit of 0.2 cancer cells mL−1. Importantly, the nanogap‐rich Au NW sensor can diagnose gastric and breast cancer tissues accurately. The nanogap‐rich Au NW sensors show strong SERS signals only in the presence of tumor tissues excised from 16 tumor‐bearing mice, while negligible signals in the presence of heated tumor tissues or normal tissues. It is anticipated that nanogap‐rich Au NW SERS sensors can be used for a universal cancer diagnosis and further biomedical applications including a diverse biomarker sensing.

Abstract 746: TaqMan Rare Mutation Assays targeting the TERT promoter region

Abstract The enzyme Telomerase maintains telomeres at the ends of chromosomes. The Telomerase Reverse Transcriptase (TERT) gene codes for the enzyme’s catalytic domain and is not expressed in normal somatic cells. As a consequence, normal cells acquire senescence by shortening of their telomeres during cell division and eventually undergo apoptosis. In contrast to normal somatic cells, expression of TERT is reinstated in cancer cells causing escape from senescence and apoptosis by maintaining the telomeres. It has recently been shown that mutations in the TERT promoter region play a key role in regulating and reinstating TERT expression. 90% of cancers carry a mutation in the TERT promoter region. Mutations like C228T and C250T create new binding site for the E26 transformation-specific (ETS) transcription factor that regulates TERT expression. Experimental evidence showed that the ETS factor GA-binding protein, alpha subunit (GABPA) binds to the de novo ETS motif and activates TERT transcription in cancer cells. We undertook a project designing TaqMan Rare Mutation assays addressing mutations in the TERT promoter. These assays are TaqMan SNP Genotyping assays that are optimized for use in digital PCR with the Applied Biosystems QuantStudio 3D. In digital PCR, partitioning the sample into many individual reaction wells facilitates detection and quantification of rare mutant alleles. TaqMan SNP Genotyping Assays ensure reliable discrimination of mutant and wild-type allele. This will enable easy and sensitive detection of TERT promoter mutations in cancer research samples. These assays are suitable for detection in liquid biopsy applications with cell free DNA (cfDNA). Assay design proved to be challenging due to high GC content and repetitive elements in this region of the TERT gene and required varying both the assay design and experimental cycling conditions. Template for wet-lab testing was synthetic plasmid carrying the mutation spiked into wild-type genomic DNA and wild-type genomic DNA control. The result was a TaqMan Rare Mutation assay detecting the C228T mutation in the TERT promoter region. We showed that designing TaqMan Rare Mutation Assays for digital PCR is feasible for the challenging TERT promoter region. Work addressing additional mutations in this region is ongoing. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Marion Laig, Kamini Varma, Vidya Venkatesh. TaqMan Rare Mutation Assays targeting the TERT promoter region [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 746. doi:10.1158/1538-7445.AM2017-746

Abstract LB-142: Identification of STK33 as a cancer stemness kinase and regulator of Nanog function

Abstract In recent years evidence has accumulated in support of the cancer stem cell (CSC) model in cancer chemotherapy resistance, highlighting the urgency and necessity of identifying CSC targets for developing novel cancer therapeutics. A number of studies have suggested that Nanog is a crucial factor that can confer stemness properties to a portion of the heterogeneous cancer cell population. Although latent in normal somatic cells, aberrant expression of Nanog has been reported in many types of human cancers. Importantly, the expression levels of Nanog are often positively correlated with treatment resistance and poor survival of cancer patients. Various studies have shown that upregulation of Nanog expression enhances the tumorigenicity both in vivo and in vitro whereas repression or ablation of Nanog inhibits tumor initiation. Thus, expression of the stemness factor Nanog is linked to tumor progression, therapeutic resistance, relapse and metastasis. However, Nanog is considered a non-druggable target. Here we provide data to support a role for STK33 (Serine Threonine Kinase 33) as a novel regulator of Nanog and as a potential therapeutic target. Ectopic STK33 expression promotes stemness phenotypes of cancer cells and increases expression levels of CSC drivers including Nanog, KLF4, and SOX2. On the other hand, knockdown of STK33 inhibits expression of Nanog and results in a reduction of the stemness phenotype. STK33 directly interacts with Nanog and appears to promote its stabilization through phosphorylation, resulting in increased Nanog transcriptional activity. Moreover, BBI-503 (Amcasertib), a first-in-class cancer stemness inhibitor, potently inhibits STK33, which led to inhibition of phosphorylation of Nanog. Collectively, our data demonstrate STK33 is a critical element in the signaling network that governs the stemness of cancer cells, and as a promising therapeutic target for cancer. Citation Format: Susan L. Tran, Yudai Furuta, Chen Zhu, Ao Yang, Xiangao Sun, Harry A. Rogoff, Chiang J. Li. Identification of STK33 as a cancer stemness kinase and regulator of Nanog function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-142. doi:10.1158/1538-7445.AM2017-LB-142

Licochalcone A specifically induces cell death in glioma stem cells via mitochondrial dysfunction.

Glioblastoma multiforme is the most malignant primary intrinsic brain tumor. Glioma stem cells (GSCs) are associated with chemoradiotherapy resistance and the recurrence of glioblastomas after conventional therapy. The targeting of GSCs is potentially an effective treatment for the long‐term survival of glioblastoma patients. Licochalcone A, a natural chalconoid from licorice root, exerts anticancer effects; however, its effect on GSCs remains unknown. We found that Licochalcone A induced massive caspase‐dependent death in GSCs but not in differentiated GSCs nor normal somatic and neural stem cells. Prior to cell death, Licochalcone A caused mitochondrial fragmentation and reduced the membrane potential and ATP production in GSCs. Thus, Licochalcone A induces mitochondrial dysfunction and shows promise as an anticancer stem cell drug.

Alternative Chk1-independent S/M checkpoint in somatic cells that prevents premature mitotic entry.

Genomic instability is the hallmark of cancer. Checkpoint kinase-1 (Chk1) is required for cell cycle delay after DNA damage or blocked DNA replication. Chk1-depleted tumor cells undergo premature mitosis and apoptosis. Here we analyzed the depletion of Chk1 in normal somatic cells in the absence of DNA damage in order to investigate alternative cell cycle checkpoint mechanism(s). By means of adenoviruses, flow cytometry, immunofluorescence and Western blotting, Chk1-depleted mouse embryonic fibroblasts (MEFs) were investigated. Chk1-/- MEFs arrested at the S/G2 boundary of the cell cycle with decreased protein levels of many cell cycle key players. Cyclin B1 was predominantly cytoplasmic. Interestingly, overexpression of nuclear dominant Cyclin B1 leads to nuclear translocation and premature mitosis. Chk1-/- MEFs exhibited the absence of double-strand breaks, yet cells showed delayed DNA damage recovery with pan-nuclear immunostaining pattern of Histone H2AX. Activation of this checkpoint would elicit a senescent-like phenotype. Taken together, our elaborated data revealed the existence of an additional S/M checkpoint functioning via γH2AX signaling and cytoplasmic retention of Cyclin B1 in somatic cells.