Junko Sakurai

Chemical Genomics Identifies the Unfolded Protein Response as a Target for Selective Cancer Cell Killing during Glucose Deprivation

Glucose deprivation, a cell condition that occurs in solid tumors, activates the unfolded protein response (UPR). A key feature of the UPR is the transcription program activation, which allows the cell to survive under stress conditions. Here, we show that the UPR transcription program is disrupted by the antidiabetic biguanides metformin, buformin, and phenformin depending on cellular glucose availability. These drugs inhibit production of the UPR transcription activators XBP1 and ATF4 and induce massive cell death during glucose deprivation as did the antitumor macrocyclic compound versipelostatin. Gene expression profiling shows remarkable similarity in the modes of action of biguanides and versipelostatin determined by the broad range of glucose deprivation-inducible genes. Importantly, during glucose deprivation, most of the biguanide suppression genes overlap with the genes induced by tunicamycin, a chemical UPR inducer. Gene expression profiling also identifies drug-driven signatures as a tool for discovering pharmacologic UPR modulators. Our findings show that disrupting the UPR during glucose deprivation could be an attractive approach for selective cancer cell killing and could provide a chemical genomic basis for developing UPR-targeting drugs against solid tumors.

A germ-line insertion in the Birt-Hogg-Dubé (BHD) gene gives rise to the Nihon rat model of inherited renal cancer

A rat model of hereditary renal carcinoma (RC) was found in a rat colony of the Sprague–Dawley strain in Japan and named the “Nihon” rat. In heterozygotes, RCs, predominantly the clear cell type, develop from early preneoplastic lesions, which began to appear as early as 3 weeks of age, to adenocarcinomas by the age of 6 months. The Nihon rat is an example of a Mendelian dominantly inherited predisposition for development of RCs like the Eker (Tsc2 gene mutant) rat. We have previously shown that the Nihon mutation was tightly linked to genes that are located on the distal part of rat chromosome 10. The order of the genes is the Eker (Tsc2 gene (human 16p13.3)–Il3 gene–Nihon gene–Llgl1 locus– Myhse gene. We now describe a germ-line mutation in the Birt–Hogg–Dubé gene (Bhd) (human 17p11.2) caused by the insertion of a single nucleotide in the Nihon rat, resulting in a frameshift and producing a stop codon 26 aa downstream. We found that the homozygous mutant condition was lethal at an early stage of fetal life in the rat. We detected a high frequency of loss of heterozygosity (LOH) in primary RCs (10/11) at the Bhd locus and found a point mutation (nonsense) in one LOH-negative case, fitting Knudsons “two-hit” model. The Nihon rat may therefore provide insights into a tumor-suppressor gene that is related to renal carcinogenesis and an animal model of human BHD syndrome.

Mitochondria regulate the unfolded protein response leading to cancer cell survival under glucose deprivation conditions

Cancer cells consume large amounts of glucose because of their specific metabolic pathway. However, cancer cells exist in tumor tissue where glucose is insufficient. To survive, cancer cells likely have the mechanism to elude their glucose addiction. Here we show that functional mitochondria are essential if cancer cells are to avoid glucose addiction. Cancer cells with dysfunctional mitochondria, such as mitochondrial DNA‐deficient ρ0 cells and electron transport chain blocker‐treated cells, were highly sensitive to glucose deprivation. Our data demonstrated that this sensitization was associated with failure of the unfolded protein response (UPR), an adaptive response mediated by the endoplasmic reticulum (ER). This study suggests a link between mitochondria and the ER during the UPR under glucose deprivation conditions and that mitochondria govern cell fate, not only through ATP production and apoptosis regulation, but also through modulating the UPR for cell survival.

Preventing the unfolded protein response via aberrant activation of 4E‐binding protein 1 by versipelostatin

We recently isolated a macrocyclic compound, versipelostatin (VST), that exerts in vivo antitumor activity. VST shows unique, selective cytotoxicity to glucose‐deprived tumor cells by preventing the unfolded protein response (UPR). Here we show that eukaryotic initiation factor 4E‐binding protein 1 (4E‐BP1), a negative regulator of eukaryotic initiation factor 4E‐mediated protein translation, plays a role in the UPR‐inhibitory action of VST. Indeed, 4E‐BP1 is aberrantly activated by VST. This activation occurs specifically during glucose deprivation and results in profound translation repression and prevents induction of the typical UPR markers glucose‐regulated protein (GRP) 78 and activating transcription factor (ATF) 4. Our overexpression and knockdown experiments showed that 4E‐BP1 can regulate GRP78 and ATF4 expression. These mechanisms appear to be specific for VST. By contrast, rapamycin, which activates 4E‐BP1 regardless of cellular glucose availability, has only marginal effects on the expression of GRP78 and ATF4. Our present findings demonstrate that aberrant 4E‐BP1 activation can contribute to UPR preventing by VST, possibly through a mechanism that does not operate in rapamycin‐treated cells. (Cancer Sci 2009; 100: 327–333)

A Novel Renal Carcinoma Predisposing Gene of the Nihon Rat Maps on Chromosome 10

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Spra‐gue‐Dawley (SD) strain in Japan, and named the “Nihon” rat in 2000. This study was designed to map the RC susceptibility gene in the Nihon rat using 113 backcross annuals. Our present data clearly show that the Nihon gene is genetically linked to interleukin‐3 (IL3) gene (χ2=93.6, Lod score=25.16), lethal (2) giant larvae (LLGL1) locus (χ2=109.0, Lod score=31.56) and myosin heavy chain, embryonic skeletal muscle (MYHSE) gene (χ2=90.6, Lod score=23.87), which are located on the distal part of rat chromosome 10. The order of the genes is the Eker (Tsc2) gene (located on the proximal part of rat chromosome 10; human chromosome 16p 13.3)–21.3 cM–IL3 gene (human 5q23‐31)–4.4 cM–Nihon gene–0.9 cM–LLGL1 locus (human 17p11.2)‐4.4 cM–MYHSE gene (human 17pl3.1). We also detected loss of the wild‐type allele at the MYHSE locus, fitting Knudsons “two hit” model. Thus, the Nihon rat should have a mutation of a novel tumor suppressor gene related to renal carcinogenesis.

Activation of Cyp1a1 and Cyp1a2 genes in adult mouse hepatocytes in primary culture.

Expression of Cyp1a1 and Cyp1a2 genes was investigated in adult C57BL/6NCrj mouse hepatocytes in primary culture for up to 5 days. When the cells were cultivated as monolayers on collagen‐coated dishes, CYP1A1 mRNA species were prominently induced after treatment with 3‐methylcholanthrene (MCA) throughout the observation period. Substantial induction of CYP1A2 mRNA by MCA was also observed at day 1 of cultivation, followed by a decrease to very low levels thereafter. In contrast, when cultivated on non‐coated dishes, the hepatocytes formed multicellular aggregates (spheroids) and prominent induction of both mRNA species was found for up to 5 days. Constitutive expression of CYP1A2 mRNA in spheroid culture was maintained throughout the observation period, whereas that in monolayer culture decreased rapidly. The time‐course of the induced CYP1A2 mRNA amounts after the treatment with MCA or 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) followed the same pattern as that of CYP1A1 mRNA. Expressed amounts of CYP1A1 or CYP1A2 mRNA in spheroid culture were higher than or similar to the levels in the case of in vivo production, respectively. Induction of both mRNA species was also observed in hepatocytes from nonresponsive DBA/2NCrj mouse in spheroid culture, but the expressed amount after MCA treatment was far smaller than for C57BL/6NCrj cells, despite equivalent expression in the two strains after TCDD. Activities of aryl hydrocarbon hydroxylase (AHH) and acetanilide 4‐hydroxylase (AAH) were elevated with either type of cultivation after treatment with MCA or TCDD, Ratios of AAH to AHH were not changed between the two cultures after 24 h treatment. However, the ratios in spheroid culture after 48 h treatment increased, whereas they did not change in monolayer culture. The present observations indicate that the spheroid culture is more suitable than the monolayer system for studying the mechanism of Cyp1a2 gene expression in adult mouse hepatocytes.

Natural history of the Nihon rat model of BHD.

Hereditary cancer was first described in the rat by Eker and Mossige in 1954 in Oslo. The Eker rat model of hereditary renal carcinoma (RC) was the first example of a Mendelian dominantly inherited predisposition to a specific cancer in an experimental animal, and has been contributing to the elucidation of renal carcinogenesis. Recently, we found a second hereditary RC model in the Sprague-Dawley (SD) rat, in Japan in 2000, which was named the Nihon rat. The Nihon rat is also an example of a Mendelian dominantly inherited predisposition for development of RCs like the Eker rat, which are predominantly of the clear cell type (this type represents approximately 75 % of human RCC), and develop from earlier preneoplastic lesions than the Eker rat. We performed a genetic linkage analysis of the Nihon rat using 113 backcross animals, and found that the Nihon mutation was tightly linked to genes, which are located on the distal part of rat chromosome 10. Finally, we identified a germline mutation in the Birt-Hogg-Dubé gene (Bhd) (rat chromosome 10, human chromosome 17p11.2) caused by the insertion of a single nucleotide in the Nihon rat gene sequence, resulting in a frame shift and producing a stop codon 26 amino acids downstream. Thus, the Nihon rat will contribute to understanding the BHD gene function and renal carcinogenesis.

Compound C Prevents the Unfolded Protein Response during Glucose Deprivation through a Mechanism Independent of AMPK and BMP Signaling

Inhibiting the unfolded protein response (UPR) can be a therapeutic approach, especially for targeting the tumor microenvironment. Here, we show that compound C (also known as dorsomorphin), a small-molecule inhibitor of AMP-activated protein kinase (AMPK) and bone morphogenetic protein (BMP) signaling, inhibit the UPR-induced transcription program depending on the glucose deprivation conditions. We found that compound C prevented UPR marker glucose-regulated protein 78 (GRP78) accumulation and exerted enhanced cytotoxicity during glucose deprivation. Gene expression profiling, together with biochemical analysis, revealed that compound C had a unique mode of action to suppress the transcriptional activation of UPR-targeted genes, as compared with the classic UPR inhibitors versipelostatin and biguanides. Surprisingly, the UPR-inhibiting activity of compound C was not associated with either AMPK or BMP signaling inhibition. We further found that combination treatments of compound C and the classic UPR inhibitors resulted in synergistic cell death with UPR suppression during glucose deprivation. Our findings demonstrate that compound C could be a unique tool for developing a UPR-targeted antitumor therapy.

Hyperactivation of 4E-binding protein 1 as a mediator of biguanide-induced cytotoxicity during glucose deprivation

Biguanides, including metformin, buformin, and phenformin, are potential antitumorigenic agents and induce cell death during glucose deprivation, a cell condition that occurs in the tumor microenvironment. Here, we show that this selective killing of glucose-deprived cells is coupled with hyperactivation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of translation initiation. We found, in fact, that the 4E-BP1 hyperactivation led to failure of the unfolded protein response (UPR), an endoplasmic reticulum–originated stress signaling pathway for cell survival. We also found that the 4E-BP1–mediated UPR inhibition occurred through a strong inhibition of the mTOR signaling pathway, a proven antitumor target. Importantly, the 4E-BP1 hyperactivation can be also seen in xenografted cancer cells through an in vivo biguanide treatment. Our findings indicate that antitumor action of biguanides can be mediated by 4E-BP1 hyperactivation, which results in UPR inhibition and selective cell killing when glucose is withdrawn. Mol Cancer Ther; 11(5); 1082–91. ©2012 AACR.

Altered regulation of Cyp1a-1 gene expression during cultivation of mouse hepatocytes in primary culture

Alterations in Cyp1a-1 gene expression in adult C57BL/6 mouse hepatocytes were followed after transferring them to primary culture during the initial 5 days. Changing the medium to a fresh one was associated with considerable amounts of Cyp1a-1 gene mRNA with a peak at around 6 hr after the medium change, followed by a decrease to negligible levels 24 hr later. Treatment of hepatocytes with cycloheximide increased the medium change-associated mRNA expression, the levels being equivalent to those observed after treatment with 3.2-25.6 nM 3-methylcholanthrene plus cycloheximide. With increasing length of culture period, cycloheximide-aided enhancement of the medium change-associated mRNA transcription increased. Although the chemical alone did not induce Cyp1a-1 gene transcripts in hepatocytes at day 1 or 2 of cultivation, for which medium had been changed 24 hr previously, prominent induction of transcripts was evident at later periods, the levels being elevated in accordance with length of time in culture. To examine whether or not the mRNA transcribed under these culture conditions was translatable, the cells were treated with actinomycin D after washing out the cycloheximide, in order to inhibit degradation of the generated mRNA (Nemoto N and Sakurai J, Carcinogenesis 12: 2115-2121, 1991). After these procedures significant elevation of aryl hydrocarbon hydroxylase activity was observed in hepatocytes, the rise being well correlated with elevated levels of mRNA transcripts. The observations suggest that the Cyp1a-1 gene might be expressed at low levels during the initial phase of cultivation of mouse hepatocytes in primary culture. Whether this expression might be essential for mouse hepatocytes to adapt to culture conditions is unclear. The findings do suggest, however, that superinducibility of the gene expression after cycloheximide treatment might be a result of a regulatory mechanism operating after adaptation to culture.