Satomi Tsukahara

Dominant-negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S-S dependent homodimerization

Breast cancer resistance protein (BCRP) is a half‐molecule ABC transporter highly expressed in mitoxantrone‐resistant cells. In our study we established PA317 transfectants expressing Myc‐tagged BCRP (MycBCRP) or HA‐tagged BCRP (HABCRP). The exogenous BCRP protein migrated as a 70‐kDa protein in SDS‐PAGE under reducing condition, but migrated as a 140‐kDa complex in the absence of reducing agents. The 140‐kDa BCRP complex was heat‐stable but dissociated into 70‐kDa BCRP with the addition of 2‐mercaptoethanol. The 140‐kDa BCRP complex was immunoprecipitated with anti‐Myc antibody from the lysates of PA317 cells double‐transfected with MycBCRP and HABCRP. The 140‐kDa complex reacted with anti‐HA and anti‐BCRP antibodies and after the addition of reducing agents, a 70‐kDa protein reacting with anti‐Myc, anti‐HA and anti‐BCRP antibodies was detected. These results clearly indicate that BCRP forms a homodimer bridged by disulfide bonds. To assess the possible dominant‐negative inhibition of BCRP drug efflux pump, various mutant BCRP cDNAs were isolated by PCR mutagenesis. First, mutant BCRP cDNAs were introduced to parental PA317 cells and tested for their function as drug‐resistance genes. Next, inactive BCRP cDNA clones were introduced to MycBCRP‐transfected cells and tested for the ability to lower drug resistance. Among the 8 inactive mutant cDNA clones tested, HABCRP cDNA clone 15 with an amino acid change from Leu to Pro at residue 554 in the fifth transmembrane domain of BCRP partially reversed the drug resistance of MycBCRP‐transfected cells. These results suggest that homodimer formation is essential for BCRP drug resistance, implicating this dominant‐negative inhibition as a new strategy to circumvent drug resistance.

Phytoestrogens/Flavonoids Reverse Breast Cancer Resistance Protein/ABCG2-Mediated Multidrug Resistance

Breast cancer resistance protein (BCRP), also called ABCG2, confers resistance to anticancer agents such as 7-ethyl-10-hydroxycamptothecin (SN-38), mitoxantrone, and topotecan. We found previously that sulfated estrogens are physiologic substrates of BCRP. Flavonoids with weak estrogenic activities are called phytoestrogens. In this study, we show that phytoestrogens/flavonoids, such as genistein, naringenin, acacetin, and kaempferol, potentiated the cytotoxicity of SN-38 and mitoxantrone in BCRP-transduced K562 (K562/BCRP) cells. Some glycosylated flavonoids, such as naringenin-7-glucoside, also effectively inhibited BCRP. These flavonoids showed marginal effect on the drug sensitivity of K562 cells. Genistein and naringenin reversed neither P-glycoprotein-mediated vincristine resistance nor multidrug resistance-related protein 1-mediated VP-16 resistance. Genistein and naringenin increased cellular accumulation of topotecan in K562/BCRP cells. K562/BCRP cells also accumulated less [3H]genistein than K562 cells. [3H]genistein transport in the basal-to-apical direction was greater in BCRP-transduced LLC-PK1 (LLC/BCRP) cells, which express exogenous BCRP in the apical membrane, than in parental cells. Fumitremorgin C abolished the increased transport of [3H]genistein in LLC/BCRP cells compared with parental cells. TLC analysis revealed that genistein was transported in its native form but not in its metabolized form. These results suggest that genistein is among the natural substrates of BCRP and competitively inhibits BCRP-mediated drug efflux. The results have two important clinical implications: (a) flavonoids and glycosylated flavonoids may be useful in overcoming BCRP-mediated drug resistance in tumor cells; and (b) coadministration of flavonoids with BCRP-substrate antitumor agents may alter the pharmacokinetics and consequently increase the toxicity of specific antitumor agents in cancer patients.

Inhibition of the mitogen-activated protein kinase pathway results in the down-regulation of P-glycoprotein

The multidrug resistance gene 1 (MDR1) product, P-glycoprotein (P-gp), pumps out a variety of anticancer agents from the cell, including anthracyclines, Vinca alkaloids, and taxanes. The expression of P-gp therefore confers resistance to these anticancer agents. In our present study, we found that FTI-277 (a farnesyltransferase inhibitor), U0126 [an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)], and 17-allylamino-17-demethoxygeldanamycin (an inhibitor of heat shock protein 90) reduced the endogenous expression levels of P-gp in the human colorectal cancer cells, HCT-15 and SW620-14. In contrast, inhibitors of phosphatidylinositol 3-OH kinase, mammalian target of rapamycin, p38 mitogen-activated protein kinase, and c-Jun NH2-terminal kinase did not affect P-gp expression in these cells. We further found that U0126 down-regulated exogenous P-gp expression in the MDR1-transduced human breast cancer cells, MCF-7/MDR and MDA-MB-231/MDR. However, the MDR1 mRNA levels in these cells were unaffected by this treatment. PD98059 (a MEK inhibitor), ERK small interfering RNA, and p90 ribosomal S6 kinase (RSK) small interfering RNA also suppressed P-gp expression. Conversely, epidermal growth factor and basic fibroblast growth factor enhanced P-gp expression, but the MDR1 mRNA levels were unchanged in epidermal growth factor–stimulated cells. Pulse-chase analysis revealed that U0126 promoted P-gp degradation but did not affect the biosynthesis of this gene product. The pretreatment of cells with U0126 enhanced the paclitaxel-induced cleavage of poly(ADP-ribose) polymerase and paclitaxel sensitivity. Furthermore, U0126-treated cells showed high levels of rhodamine123 uptake. Hence, our present data show that inhibition of the MEK-ERK-RSK pathway down-regulates P-gp expression levels and diminishes the cellular multidrug resistance. [Mol Cancer Ther 2007;6(7):2092–2102]

Estrone and 17β‐Estradiol Reverse Breast Cancer Resistance Protein‐mediated Multidrug Resistance

Breast cancer resistance protein (BCRP), an adenosine triphosphate‐binding cassette transporter, confers resistance to a series of anticancer reagents, including mitoxantrone, SN‐38 and topotecan. In the present study, we found that estrone and l7β‐estradiol potentiated the cytotoxicity of mitoxantrone, SN‐38 and topotecan in BCRP‐transduced K562 cells (K562/BCRP). These estrogens showed only a marginal effect, or none, in parental K562 cells. Estrone and 17β‐estradiol increased the cellular accumulation of topotecan in K562/BCRP cells, but not in K562 cells, suggesting that these estrogens inhibit the BCRP‐mediated drug efflux and overcome drug resistance.

Breast cancer resistance protein: molecular target for anticancer drug resistance and pharmacokinetics/pharmacodynamics.

Breast cancer resistance protein (BCRP) is a half‐molecule ATP‐binding cassette transporter that forms a functional homodimer and pumps out various anticancer agents, such as 7‐ethyl‐10‐hydroxycamptothecin, topotecan, mitoxantrone and flavopiridol, from cells. Estrogens, such as estrone and 17β‐estradiol, have been found to restore drug sensitivity levels in BCRP‐transduced cells by increasing the cellular accumulation of such agents. Furthermore, synthetic estrogens, tamoxifen derivatives and phytoestrogens/flavonoids have now been identified that can effectively circumvent BCRP‐mediated drug resistance. Transcellular transport experiments have shown that BCRP transports sulfated estrogens and various sulfated steroidal compounds, but not free estrogens. The kinase inhibitor gefitinib inhibited the transporter function of BCRP and reversed BCRP‐mediated drug resistance both in vitro and in vivo. BCRP‐transduced human epidermoid carcinoma A431 (A431/BCRP) and BCRP‐transduced human non‐small cell lung cancer PC‐9 (PC‐9/BCRP) cells showed gefitinib resistance. Physiological concentrations of estrogens (10–100 pM) reduced BCRP protein expression without affecting its mRNA levels. Two functional polymorphisms of the BCRP gene have been identified. The C376T (Q126Stop) polymorphism has a dramatic phenotype as active BCRP protein cannot be expressed from a C376T allele. The C421A (Q141K) polymorphism is also significant as Q141K‐BCRP‐transfected cells show markedly low protein expression levels and low‐level drug resistance. Hence, individuals with C376T or C421A polymorphisms may express low levels of BCRP or none at all, resulting in hypersensitivity of normal cells to BCRP‐substrate anticancer agents. In summary, both modulators of BCRP and functional single nucleotide polymorphisms within the BCRP gene affect the transporter function of the protein and thus can modulate drug sensitivity and substrate pharmacokinetics and pharmacodynamics in affected cells and individuals. (Cancer Sci 2005; 96: 457–465)

Single amino acid substitutions in the transmembrane domains of breast cancer resistance protein (BCRP) alter cross resistance patterns in transfectants

Breast cancer resistance protein (BCRP) is a member of ATP‐binding cassette transporters that has an N‐terminal ATP binding domain and a C‐terminal transmembrane domain (TM). Expression of wild‐type BCRP confers resistance to multiple chemotherapeutic agents such as mitoxantrone, SN‐38 and topotecan, but not to doxorubicin. We made 32 BCRP mutants with an amino acid substitution in the TMs (7 E446‐mutants in TM2, 15 R482‐mutants in TM3, 4 N557‐mutants in TM5 and 6 H630‐mutants in TM6) and examined the effect of the substitutions on cellular drug resistance. PA317 cells transfected with any one of the 7 E446‐mutant BCRP cDNAs did not show drug resistance. Cells transfected with any one of the 13 R482X2‐BCRP cDNAs (X2 = N, C, M, S, T, V, A, G, E, W, D, Q and H, but not Y and K) showed higher resistance to mitoxantrone and doxorubicin than the wild‐type BCRP‐transfected cells. Cells transfected with N557D‐BCRP cDNA showed similar resistance to mitoxantrone but lower resistance to SN‐38 than the wild‐type BCRP‐transfected cells. Cells transfected with N557E‐, H630E‐ or H630L‐BCRP cDNA showed similar degrees of resistance to mitoxantrone and SN‐38. Estrone and fumitremorgin C reversed the drug resistance of cells transfected with R482‐, N557‐ or H630‐mutant BCRP cDNA. Cells transfected with R482G‐ or R482S‐BCRP cDNA showed less intracellular accumulation of [3H]mitoxantrone than the wild‐type BCRP‐transfected cells. These results suggest that E446 in TM2, R482 in TM3, N557 in TM5 and H630 in TM6 play important roles in drug recognition of BCRP.

Estrogen-mediated post transcriptional down-regulation of P-glycoprotein in MDR1-transduced human breast cancer cells

The human multidrug resistance gene 1 (MDR1) encodes the plasma membrane P‐glycoprotein (P‐gp/ABCB1) that functions as an efflux pump for various anticancer agents. We recently reported that estrogens down‐regulate the expression of breast cancer resistance protein (BCRP/ABCG2). In our present study we demonstrate that estrogens also down‐regulate P‐gp expression in the MDR1‐transduced, estrogen receptor α (ER‐α)‐positive human breast cancer cells, MCF‐7/MDR and T‐47D/MDR. The P‐gp expression levels in MCF‐7/MDR cells treated with 100 pM estradiol were found to be 10–20‐fold lower than the levels in these same cells that were cultured without estradiol. In contrast, estradiol did not affect the P‐gp expression levels in the ER‐α‐negative cancer cells, MDA‐MB‐231/MDR and NCI/ADR‐RES. Estrone and diethylstilbestrol were also found to down‐regulate P‐gp in MCF‐7/MDR cells, but progesterone treatment did not produce this effect. Tamoxifen reversed the estradiol‐mediated down‐regulation of P‐gp in MCF‐7/MDR cells, suggesting that ER‐α activity is necessary for the effects of estradiol upon P‐gp. However, estradiol was found not to alter the MDR1 transcript levels in either MCF‐7/MDR and T‐47D/MDR cells, suggesting that post‐transcriptional mechanisms underlie its effects upon P‐gp down‐regulation. MCF‐7/MDR cells also showed eight‐fold higher sensitivity to vincristine when treated with 100 pM estradiol, than when treated with 1 pM estradiol. These results may serve to provide a better understanding of the expression control of ABC transporters, and possibly allow for the establishment of new cancer chemotherapy strategies that would control P‐gp expression in breast cancer cells and thereby increase their sensitivity to MDR1‐related anticancer agents. (Cancer Sci 2006; 97: 1198–1204)

A Novel Endoplasmic Reticulum Export Signal PROLINE AT THE +2-POSITION FROM THE SIGNAL PEPTIDE CLEAVAGE SITE

NUCB1 (nucleobindin 1) is a Golgi-localized soluble protein with a signal peptide and multiple functional domains. We reported recently that NUCB1 is a negative regulator of the unfolded protein response that activates various endoplasmic reticulum (ER)-originating signaling pathways. In that report, we also showed that Golgi localization of NUCB1 was essential to regulate the unfolded protein response. However, the localization mechanism of NUCB1 is still unknown. Here, we report that the proline residue at the +2-position (Pro+2) from the signal peptide cleavage site is the determinant of NUCB1 protein export from the ER and subsequent transport to the Golgi. Fusion of the N-terminal amino acids 1–35 peptide region, including both signal peptide (amino acids 1–26) and Pro+2, was sufficient for enhanced green fluorescent protein to localize in the Golgi, whereas single amino acid mutation of Pro+2 resulted in defective export from the ER without affecting the protein maturation process. Furthermore, we demonstrated that Pro+2 was important for the enhanced green fluorescent protein fusion protein to concentrate at a transport vesicle formation site within the ER, often termed the ER exit site. Interestingly, such a Pro+2 has also been functionally conserved in other Golgi-localized soluble proteins, Cab45 (Ca2+-binding protein of 45 kDa), reticulocalbin 1, and calumenin. Our findings indicate that Pro+2 can function as a novel ER export signal of some Golgi proteins.

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)

The identification of two germ-line mutations in the human breast cancer resistance protein gene that result in the expression of a low/non-functional protein

PurposeWe examined the effects of the nine nonsynonymous germ-line mutations/SNPs in the breast cancer resistance protein (BCRP/ABCG2) gene on the expression and function of the protein.Materials and MethodsWe generated cDNAs for each of these mutants (G151T, C458T, C496G, A616C, T623C, T742C, T1291C, A1768T, and G1858A BCRP) and compared the effects of their exogenous expression in PA317 cells with a wild-type control.ResultsPA/F208S cells (T623C BCRP-transfectants) expressed marginal levels of a BCRP protein species (65kDa), which is slightly smaller than wild-type (70kDa), but this mutant did not appear on the cell surface or confer drug resistance. PA/F431L cells (T1291C BCRP-transfectants) were found to express both 70kDa and 65kDa BCRP protein products. In addition, although PA/F431L cells expressed 70kDa BCRP at comparable levels to PA/WT cells, they showed only marginal resistance to SN-38. PA/T153M cells (C458T BCRP-transfectants) and PA/D620N cells (G1858A BCRP-transfectants) expressed lower amounts of BCRP and showed lower levels of resistance to SN-38 compared with PA/WT cells.ConclusionsWe have shown that T623C BCRP encodes a non-functional BCRP and that T1291C BCRP encodes a low-functional BCRP. Hence, these mutations may affect the pharmacokinetics of BCRP substrates in patients harboring these alleles.