Cancer relapse occurs with substantial frequency even after treatment with curative intent. Here we studied drug-tolerant colonies (DTCs), which are subpopulations of cancer cells that survive in the presence of drugs. Proteomic characterization of DTCs identified stemness- and epithelial-dominant subpopulations, but functional screening suggested that DTC formation was regulated at the transcriptional level independent from protein expression patterns. We consistently found that α-amanitin, an RNA polymerase II (RNAPII) inhibitor, effectively inhibited DTCs by suppressing TAF15 expression, which binds to RNA to modulate transcription and RNA processing. Sequential administration of α-amanitin and cisplatin extended overall survival in a cancer-relapse mouse model, namely peritonitis carcinomatosa. Therefore, post-treatment cancer relapse may occur through non-distinct subpopulations and may be effectively prevented by α-amanitin to disrupt transcriptional machinery, including TAF15.

SUMMARYSeveral reports have demonstrated a close correlation between plasma atrial natriuretic peptide (ANP) concentration and atrial pressure in stable heart diseases. However, few studies have investigated whether plasma ANP concentration is a noninvasive indicator of hemodynamic parameters during the treatment of heart failure. Thus, we have studied the relationship between peripheral plasma ANP concentration and concurrent hemodynamic variables during the treatment of heart failure, and, in order to determine whether secretion of ANP is stimulated in this disease condition, we compared the plasma ANP concentration in the pulmonary artery with that in the peripheral veins.Studies were performed in each of 9 patients with acute heart failure due to myocardial infarction (Group A) or chronic heart failure (Group B), who were matched as closely as possible for treatment, age, sex and cardiac output. In group A, no significant correlation was found between plasma ANP levels and any measured hemodynamic variables. In group B, peripheral plasma ANP concentrations were significantly correlated with left atrial pressure (r=0.82, p<0.01), but not with right atrial pressure (r=0.56, p>0.05). Furthermore, in group B ANP levels in pulmonary arterial plasma were consistently higher than those in peripheral venous plasma, whereas in group A the opposite was observed in expired cases.These results suggest that measurement of peripheral plasma ANP is a useful noninvasive method for estimating left atrial pressure during the treatment of chronic heart failure. However, plasma ANP concentration may not be a valid means of estimating hemodynamic parameters in acute heart failure due to myocardial infarction. In such cases, the increased secretion of ANP was not obvious, and there may be other factors , in addition to atrial pressure, that regulate cardiac secretion of ANP.

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[Background] Functional heterogeneity within cancer cell populations plays an important role in responses to genotoxic stresses caused by anticancer agents. We have characterized 2,400 colonies derived in the presence of anticancer drugs using human cancer cell lines by newly introduced colony lysate array technique (CoLA). In the present study, we attempted to identify molecular components that could induce anticancer drug-tolerance phenotypes. [Materials and Methods] Using five cancer cell lines and four anticancer agents, CoLA was produced using 2,400 drug-tolerant colonies, followed by protein expression analysis employing specific immunodetection and using a panel of primary antibodies. Transcriptional products that may be important to the formation of colonies in the presence of anticancer drugs were identified by DNA microarray. Candidate genes were examined to determine whether they were associated with the suppression of colony formation, using gene knockdown by siRNA. [Results] This study revealed that CoLA is capable of analyzing lysates from individual colonies in a quantitative manner, by a panel of protein markers. Colonies that expressed pluripotent and CSC markers tended to express low levels of epithelial proteins. However, individual protein expression levels were not well-associated with drug concentrations, suggesting that the phenotypes of drug-tolerant colonies were not necessarily induced by drugs, and emerged spontaneously. Subsequent analysis identifying molecular fractions important to drug-tolerant phenotypes revealed that transcriptional products play an important role in developing these phenotypes. DNA microarray analysis allowed for the narrowing down of 12 genes that are potentially associated with these phenotypes. A gene knockdown by siRNA identified two out of 12 candidate genes as involved in the suppressed colony formation of MCF7 human breast cancer cell line in the presence of cisplatin. [Discussion] CoLA assay revealed that a number of CSC marker-negative colonies exist in the presence of anticancer drugs, suggesting that drug-tolerant cells may not always be associated with CSCs. In the context of drug-tolerant phenotype acquisition, protein expression may merely be a handy marker, whereas transcriptional control may be more critical. Further functional analysis of candidate genes for colony suppression in the presence of anticancer drugs is necessary. Together, de novo drug-tolerance inducing factors are possibly involved in the heterogeneity of cancer cell populations. Citation Format: Kohei Kume, Satoshi Nishizuka, Miyuki Ikeda, Sawako Miura, Yuriko Wada, Shuji Fujikawa, Chihaya Maesawa, Go Wakabayashi. Analysis of cancer cell populations by isolated single colonies for the identification of drug-tolerance inducing factors. [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 4060. doi:10.1158/1538-7445.AM2013-4060

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[Background] Increasing evidences indicate that heterogeneity within cancer cell population plays a critical role in response to cytotoxic stresses including drug treatment. We characterized human cancer cell populations that formed colonies in the presence of anticancer drugs at cellular and molecular levels. [Materials and Methods] For colony formation, human cancer cells (HCT-116, HeLa, HT-29, MCF-7, and MKN-45) were sparsely disseminated (10 cells/cm2) in the presence of anticancer drugs (cisplatin, docetaxcell, gefitinib, and sorafenib). To characterize the drug-tolerant individual colonies, we established colony lysate array (CoLA), a tool used for the protein expression analysis of a single-colony, using modified “reverse-phase” protein lysate microarrays (RPAs). [Results] For in vitro modeling of cancer relapse after curative treatment, we consistently observed cell populations that form colonies under 0.1- 10 μM drug concentrations. Interestingly, the 50% colony-inhibition concentration was consistently more than 10-fold higher than that of growth-inhibition for cell-based assays. CoLA assay allows one to use a lysate from a single colony for hierarchical clustering by a panel of cancer stem cell (CSC) markers. Unlike FACS analysis, CoLA is advantageous in detection of full fractions of proteins including those in the nucleus. Thus far, we have found that MKN-45 colonies in the presence of cisplatin expressed CD44, a putative CSC marker in a concentration-dependent manner. Pre-treatment of actinomycin D (AMD), an RNA synthesis inhibitor strongly suppressed MKN-45 colony formation in the presence of cisplatin; however cycloheximide, a protein synthesis inhibitor did not affect the colony formation. [Discussion] Differences in drug concentrations between the colony-inhibition and the growth-inhibition indicate that colony formation and simple cell growth are functionally different. The CD44 expression in MKN-45 colonies in the presence of cisplatin may suggest that these colonies emerged in the presence of drugs acquired a “CSC-like” phenotype. The suppression of colony formation by AMD may also suggest that the colonies require de novo RNA synthesis for the gain of drug resistant phenotype. [Conclusion] The drug-tolerant cell populations exhibit a CSC-like phenotype and require de novo RNA synthesis for the gain of drug resistant phenotype, possibly involved in the heterogeneity of cancer cell population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4229. doi:1538-7445.AM2012-4229

Background: Cancer relapse after curative treatment is thought to originate from drug-tolerant cancer cell subpopulations. To isolate and characterize the drug-tolerant cell subpopulations, we collected individual colonies that emerged in the presence of anticancer drugs and performed proteomic profiling using reverse-phase protein arrays (RPPAs). Materials and Methods: Cancer cells (HCT116, HeLa, HT29, MCF7, and MKN45) were sparsely disseminated (10-100 cells/cm2) into the presence of anticancer drugs (cisplatin, docetaxel, gefitinib, and sorafenib) of six serially diluted concentrations on the basis of 50% colony inhibitory concentration (CI50) for each drug. A total of 2,400 individual colony lysates (20 colony lysates per drug concentration) were printed on glass slides using a dedicated microarrayer followed by immunochemical detection of 44 functionally-diverse proteins. A transcriptional profiling followed by gene ontology (GO) analysis was also performed. Results: CI50 of cisplatin and docetaxel were &gt;10 times lower than the 50% growth inhibitory concentration (GI50) in all cell lines. This indicates that colony formation is functionally different from exponential cell growth, and drug-tolerant colony initiation can be suppressed by traditional anticancer drugs with very low doses. Colony lysate arrays revealed that the colonies expressing stemness, pluripotent, and mesenchymal markers tended to show low levels of epithelial proteins. However, individual protein expression levels were not well associated with drug concentrations, suggesting that these protein phenotypes of colonies were not necessarily induced by anticancer drugs. Instead, they may have emerged spontaneously. To identify molecular fractions important to drug-tolerant phenotypes, we screened compounds inhibiting chromatin formation, transcription, and protein biosynthesis. A transcriptional inhibitor eliminated drug-tolerant colonies within a short (four-hour) temporal treatment, suggesting that putative drug-tolerance inducing factors were controlled at the transcriptional level. Analysis of cDNA microarray using drug-free and cisplatin-resistant colonies followed by GO analysis revealed that most expressed genes in cisplatin-resistant colonies were specifically associated with embryonic development. Conclusion: These findings suggest that the regulatory system at the protein level is unlikely to be directly associated with drug-tolerant colony formation. Instead, transcriptional regulation appears to play a crucial role in forming drug-tolerant colonies. Citation Format: Kohei Kume, Satoshi Nishizuka, Miyuki Ikeda, Sawako Miura, Fumitaka Endo, Katagiri Hirokatsu, Kaoru Ishida, Kei Sato, Chihaya Maesawa, Go Wakabayashi. Transcriptional and proteomic profiling of drug-tolerant cancer cell subpopulations using colonies that emerge in the presence of anticancer agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3767. doi:10.1158/1538-7445.AM2014-3767