- Email: [email protected]
A pleiotropic response associated with resistance of breast cancer cells to antineoplastic drugs and hormonal agents
TIPS -December
1988 Wol. 91
443 and genetic changes associated with multidrug resistance in addition to P170 overexpression has been an active area of research, and alterations in the levels of a number of proteins, including protein kinase Cl’, sorcin”,+cose-6phosphate dehydrogenase’* and glutathione peroxidase’3*‘4 have been identified in multidrugresistant cells. Furthermore, while analysis of
normal tissues and tumor samples has shown that some human
Philip P. Vickers, Robert B. Dixon and Kenneth H. Cowan A multidrug-resistant varknt of the human breast cancer cell line MCF-7 exhibits multiple biochemical changes including reduced estrogen receptor and increased EGF receptor levels, overexpression of the putative drug efflux pump and cross-resistance to’ carcinogens, estrogens and antiestrogens. Philip Vickers and colleagues consider the usefulness of this model to the study of clinical transition of breast cancer to a hormonally unresponsive, highly malignant state. ’ For patients with breast cancer, durable remissions may be achieved by treatment with cytotoxic drugs or hormonal agents, either singly or in combination. Unfortunately, although initial response rates to these agents are usually high, most patients even-. tually relapse. In the setting of relapse, breast cancers rapidly develop resistance to chemotherapy. Recent studies have identified a number of factors that may be involved in the development of clinical resistance. The multidrug-resistance phenotype To develop a model to study mechanisms of resistance 10 antineoplastic drurq in vitro, a number of laboratories have isolated drugresistant cell lines. Such lines are selected for resistance to single agents yet frequently display cross-resistance to a number of drugs that are dissimilar in both structure and proposed mechanism of action. Drugs associated with this multidrug-resistance phenotype include the anthracyclines, epipodophyllotoxins, vinca alkaloids and actinomycin D (Refs I and 2). Multidrug-resistant cells usually display decreased drug accumulation> , which is associated with the OverexFression of a surface glycoprotein known as P170, gp140-180, P-glycoprotein or
the mdr gene product5f6. The putative role of this protein as a drug efflux pump has recently been reviewed6. While overexpression of P170 is a common feature of multidrugresistant cell lines, this alteration may not complete!y account for the expression of multidrug resistance. Each multidrug-resistant cell line exhibits a unique pattern of cross-resistance. Although mutations in the mdr gene7 or other factors may alter the P170 protein it is unclear whether the diversity in resistance patterns can be accounted for at the level of the P170 protein. Defects in drug accumulation occur in most multidrug-resistant cell lines, tut the magnitude of these defects is often insufficient to account fclr the overall degree of drug :esiQance”. In addition, cell lines that display multidrug resistance without overexpression of P170 have been described’. Identifying biochemical
tumors express relatively high levels of P170, there is as yet no clear association between the development of clinical drug resistance and the expression of P170 in human tumors (Ref. 15 and J. A. Moscow et al., unpublished).
Biochemical changes in multidrug-resistant breast cancer cells To investigate the mechanisms associated with the development of multidrug resistance in breast cancer, our laboratory has selected an adriamycin-resistant MCF-7 human breast cancer c-l! !inc (MCF-71AdrR) which displays the multidrug-resistance 4 13.16
‘in_: I
r;llstance
pheno-
in MCF-7i
2:; ‘is ass#Jciated with a two- to three-fold decrease in drug accumulation. and a 45-fold increase in the expression of P170.
Detailed
analysis
revealed
that
MCF-7!AdrR cells have altemtions in the levels of a number ol: proteins’0,‘2-‘4*‘6 including several drug-metabolizing mzymes’3*‘7*‘” Drug-metabolizing enzymes are generally classified into phase I and phase II enzymes. Philse I .. enzymes oxialzc a wide rclrq!:: uf planar lipophilic toxins. For many carcinogens and procarcinogens (e.g. benzolalpyrene) the inter-
mediates
genevated
by
these
TABLE I. Properties of multidrug-resistant MCF-7/AdrA cells, xenobictic-resistant rat :Ipperplastic nodules, and estrogen receptor-negative breast cancer cells Cell type
Characterlstlc
L ER t EGFR f Tumorigenicity r Protein kinase C t GST-x i AHH Resistance to benzo[a]pyrene T P170
yes4
rat liver hyperplastic nodules
ER-negative breast cancer cell lines
z-negative breast cs?cczr?
ND ND :;a0 yes30 yes30 ye.9
yes ye? yesa yesz9 yesyesz’ yeszB
yes ye? NA ND vesz7 fiD ND
yes18
ND
ND
ND, not determined: NA, not applicable: ER, estrogen receptor; EGFR, EFG receptor.
TOXIN-SENSITIVE CELL
TOXIN-RESISTANT CELL
Fig. f Mxid of toxin resistance in breast cancercelfs.f. toxin; T-OH,oxygenafed foxin; TX, foxin wnjugaf@: P170, toxin efffux pimp; ER, estrogen receptor; EGFR, epkkrmalgrowfhfactctrrecepbr.Intoxin-sensitive cells (lefi) acfivity ofphasef drug-melabolizingenzymes (1)is high,leading ic7high concenfratims of thrr oxygenaled toxin producfs, which bind DNA 13); the level of estrogen ~eceprors is afsr, high. In fu~#-fesfsfa~t &Es (righ!) activityofpmst3 I dr~~-~~~ab~[~~~~ t%ZWk?s(I) iS#Ck.Ke~and t&at&p&Se HenqJRes is i#crt?ased,fr?i&ir?g tc reduce&accumula&%t and cyrfmxicity (3) of oxygenated foxin; PI 7%mediated &f/m (4) wd EGF receptor d&?&y are great& irnxeased. The roles of fbe estrogen and EGF receptors in toxic.iry are unclear.
are, paradcxicaliy, more binders ot DNA, 2Ed are more cytotoxic 17. However, the products of the phase f reactions
enzymes potent
arc also sub;s:r;ri~s of phase II drugmetabolizing enzymes: these enzymes conjugate the products of the phase I reactions with hydrophilic moieties such as glutathione or glucuronide. The resulting conjugates are readily excreted from the cell. In MCF-71AdV”cells, the activity of the anionic isozyme of the phase 11 enzyme glut Ithione-S-transkrase (GST-n) is increased 45fold’“*“. (11 addition, m~ltidrug resistance in MCF-7/A&” is associated with downregulation of the phase I enzyme arylhydrocarbon hydroxylase (AHH)‘“. The roles of GST and AHH in resistance to drugs associated with multidrug r&stance are currently under investigation; a possible mechanism is shown in Fig. 1. The alterations in drug-rn~tabo~~~ing enzymes and other proteins in MCF7lAdr” emphasize that the development of multidrug resistance in breast cancer may be associated with multiple biochemical changes.
Multidrugresistame and carcinogen resistance The biochemical ajteratians associated with wkuttidrug wsistanrp
in MCF-7/Adp, including increased expression of P170 (Ref. 4) and GST-n (Refs 23 and 16) and downreguiatian of AHH’” bear a striking similarity to alterations observed in rat premalignant hyperplastic liver nodules in the Solt-Faber model of chemical carcinogencsis16**g. Although these nodules are induced by a variety of carcinogens, they resemble multidrug-resistant cells in that they develop resistance not only to the agent used in their selection, but also to a wide range of hepatatoxins. indeed, studies indicate that rat hyper~lasti~ nodules develop resistance to a wide range of antineoplastic agen tszol while our laboratory has shown that multidrug resistance in MCF7/AdvR is associated with CTQSSresistance to the carcinogen ~en~~~~~py~ene~~.
The similarities between rat hyperplastic nodules and MCF-7! 24d2”cells raise the possibility that tumors derived from tissues with exposure to chemicalbcarcinogens may be predisposed to be resistant to anticancer agents. Clinirat evidence appears to supporl this, as tumors of the colon and lung tissues with exposure to relatively high levels of carcinogens - are USUdl~ refractory to chemotherapy in contrast, tumors derived from tissues with lOl%F Ievzit:
af exposure
to carcinogens (such as lymphomas) appear to acquire the resistance phenotype only after exposure to chemotherapy. Cross-resistance to antiestrrrgens As breast cancer may be swccessfully treated both by cytotoxic drugs and hormonal agents, we were interested in whether the development of multidrug-resistance in MCF-7/AArR resulted in any alteration in the hormonal sensitivity of the cell line. Wildtype MCF-7 cells are an excellent model system for the study of hormone-res~~nsi~~e breast cancer, as they contain receptors for both estrogen and progesterone, and are recponsive to physiological concentrations of estrogcns2’. The development of multidrug resistance in the MCF-Y’/Anr”R cell line was found to be associated with a loss of response to estrogen and cross-resistance to the antiestrogen 4-hydro~ytamo~~f~n2~” Furthermore, while wild-type MCF-7 cells require estrogen to form tumors in athymic mice, the alterations in hormonal sensitivity in MCF-71AdP have resulted in the cells being able to form tumors equally well in the absence or presence of estrogenzz. The development of hormonal ~~~en5it~vity in MCF-?/A&” is associated with loss of theestrogen
TlPS - December 2988 [Vol. 91 receptor. In contrast, this cell line displays a 100-fold increase in levels of the epidermal growth factor (EGF) receptor, and a loss of mitogenic response to EGF (Ref. 22). Other multidrug-resistant cell lines have been reported to have increased levels of EGF receptos3, although the magnitude of the change appears to be larger in MCF-7/A& R. Previous studies have shown that acute treatment of cells with cytotoxic drugs can decrease levels of the estrogen receptos4 and increase levels of the EGF recepto?‘. Although associated with cytotoxicity, these effects were rapidly reversible upon removal of drug from the medium. It remains unclear whether the mechanisms responsible for these transient alterations in receptor levels bear any relationship to the stable recaptor alterations induced in MCF7’iAdp following chronic drug treatment. A common mechanism of resistance to toxins? The finding that the development: of multidrug resistance in MCF-7/A&” is associated with multiple biochemical changes, including alterations in the expression of AHH and GST-x, as well as the loss of estrogen receptors, prompted us to examine the expression of these drug-metabolizing enzyme8 in other breast cancer cell lines with various levels of estrogen receptors. These studies revealed that the estrogen receptor-positive cell lines possessed significantly higher levels of AHH activity than the estrogen receptor-negative cell line++‘. Other studies from our laboratory have shown an inverse relationship between GST-n: activity and estrogen receptor levelsz7. These alterations are associated with resistance to both ellipticine and benzofalpyrene in estrogen receptor-negative cell line?. Protein kinase C activity has also recently been shown to bear a relationshi p to both multidrug resistance’ e2s and the estrogen rece tor status of breast cancer cells Pg. As previously described, the development ot resistance to carcinogens in rat hepatocytes and cytotoxic drugs in MCF-7/A@ is associated with a similar pattern of biochemical alterations. Our findings suggest that a number of these
445 alterations are also related to levels of estrogen receptor in breast cancer cells (Table I and Fig. 1). In addition to providing an invitro model of multidrug resistance in breast cancer, the development of hormonal insensitivity in MCF7/Adra bears analogies to the clinical progression of breast cancer. It is well established that estrogen receptor- and progesterone receptor-negative tumors have a poorer prognosis than those possessing these receptors. Furthermore, increased levels of the EGF receptor in primary breast cancer have been reported to be associated with tumors having the poorest profinosis32,33. Selection of MCF-7/A&” has therefore resulted in the progression from estrogen receptorand progesterone receplor-positive, hormone-responsive breast cancer cells to cells displaying features of the poorest prognosis breast tumors: estrogen receptorand progesterone receptor-negative, increased levels of the EGF receptor, resistance to drugs and hormonal agents, and increased tumorigenicity. q
q
0
biochemical The multiple changes associated with multidrug resistance in MCF-7/A&” define a conserved pleiotropic response to cytotoxins, and appear to be relevant to the progression of breast cancer in viva. MCF-71AdrR cells therefore represent a useful model to study both the mechanism of multidrug resistance in breast cancer and the transition of breast cancer to a hormonally unresponsive, highly malignant state. The finding that a similar pattern of gene expression occurs in a multidrug-resistant MCF-7 breast cancer cell line, rat hyperpiastic nodules and estrogen receptornegative cells suggests that similar factors regulate biochemical alterations associated with these three model systems. It is a major challenge to determine both what regulates these alterations and the role that each of the biochemical changes may play in resistance to antineoplastic drugs, chemical carcinogens and hormonal agents. References 1 Vtckers,
P. J.,
Townsend, A. 1. and
Cowan, K. H. CRC Crib Rw. Dev. Cffttcer Cttcrtrothrr
(in $-TSS?
2 Moscow, J. A. and Cowan, K. H. (1988) J. h’uff Cancer Inst. 80, 14-20 3 Skovsgaard, T. (1978) Cancer Reg. 38, 4722-4727 4 Fairchild, C. R. et al. (1987) Cancer Res. 47, 5141-5148 5 Riordan, J. R. and Ling, V. (1985) P~ffr~scol. 7&r. 28.51-75 6 Cottesman, M. M. and Pastan, I. (1988) Tresds Pharmacol.
Sci. 9, 54-58
7 Choi, K.. Chen, C-j., Kriegler, M. and Roninson, I. B. (1988) Cell 53,519-529 8 Sirotnak, F. M., Yang, C. H., Mines, L. S., Oribe, E. and Biedler, J. L. (1986) J. Ceil. Physiol. 126, 266-274 9 McGrath,.T. and Center, M. S. (1987) Biochem. Biophys. Res. Commun. 145, 1171-1176 10 Fine, R. L., Pate!, J, and Chabner, B. A. (1988) Droc. Nat1 Acnd. Sci. USA 85, 582-586 11 van der Blieck, A. M., Meyers, M. B., Riedler, J. L., Hes, E. and Borst, P. (1986) EM80 J. 5,3201-3208 12 Yeh. G. C.. Occhiointi. S. ‘1.. Cowan. K. I%, Chadner, B. x. and b&&s, C. E: (1987) Cancer Res. 47.5994-5999 13 Batist, G., Tulpule, A., Sinha, B. K., Katki, A. G., Myers, C. E. and Cowan, K. H. (1986) I. &%I. Chem. 261, 1x%4-15549 14 Kramer, R. A., Zahker, J. and Kim, J. (1988) Science 241, 694-697 15 Fojo, A. T., Ueda, K., Slamon, D. J,, Poplack. D. G., Gottesman, M. M. and Pastan, f. (1987) Proc. Natf Acad. Sci. USA 84,265-269
16 Cowan. K. H.. Batist. G.. Tuloule. A., Sinha, b. K. a&l Mye&., C.‘E. (l&i) &oc~ Nail Acnd. Sci. USA 83,9328-9332 17 Nebert, D. W. and Gelboin, H. (1986) J. Biol. Cfiem. 243, 6242-6249 18 Ivy, S. P. et al. /. Bid. Chem. (in press) 19 Fairchild, C. R. et al. (1987) Proc. Mrtl Acad. Sci. USA 84,7701-7705 20 Carr, B. (1987) Cancer RD. 47,5577-5583 21 Lippman, M: E., Bolan, G. and Huff, rZ. K. (1976) Cancer Rcs. 36, 4595-4601 22 Vickers, I’. J., Dickson, R. B.,Shoemaker, R. and Cowan, K. H. Mol. Endoerirrol. (in P=Sf 23 Meyers, M. B., Merluzzi, V. J., Spengler, B. A. and Biedler, 1. L. (1986) Proc. Nat/ Acad. Sci. USA 83,.55215525 24 Clarke, R., Morwuod, J., van den Berg, H. W., Nelson, J. and Murphy, R. F. (1986) Cancer Res. 46,611&6119 Zuckier, G. and Tritton, T. R. (1983) Erp. Cell. Res. 148, 155-161 Vickers, P. J., Dufresne, M. J. ar.d Cowan, K. H. Moi. Endocrinof. (in press) Moscow, J. A. et al. Proc. NafI Acud. Sci. USA (in press) Ferguson, P. J. and Cheng, Y-c. (1913 Cancer Res. 47,433-441 29 Borner, C., Wyss, R., Regazzi, R., Eppenberger, U. and Fabbro, D. (1987) Inf. J. Cancer 40‘344-348 30 Farber, E. (1984) Cancer Res. 44, 5436-5474 N. E., Gelmann, E. P., 31 Davidson, Lippman, M. E. and Dickson, R. B. (1987) Mot. E~docrina~, 1‘216-223 32 Brunner, N., Osborne, C. K. and SpangThomsen, M. (1987) Breast Cancer Res. Treat. 10, 229-242 33 Sainsbury, J. R. C., Farndon, J. R., Needham, G. K., Malcolm, A. J. and Harris, A. L. (1987) Lstlcet i, x398-1402
1988 Wol. 91
443 and genetic changes associated with multidrug resistance in addition to P170 overexpression has been an active area of research, and alterations in the levels of a number of proteins, including protein kinase Cl’, sorcin”,+cose-6phosphate dehydrogenase’* and glutathione peroxidase’3*‘4 have been identified in multidrugresistant cells. Furthermore, while analysis of
normal tissues and tumor samples has shown that some human
Philip P. Vickers, Robert B. Dixon and Kenneth H. Cowan A multidrug-resistant varknt of the human breast cancer cell line MCF-7 exhibits multiple biochemical changes including reduced estrogen receptor and increased EGF receptor levels, overexpression of the putative drug efflux pump and cross-resistance to’ carcinogens, estrogens and antiestrogens. Philip Vickers and colleagues consider the usefulness of this model to the study of clinical transition of breast cancer to a hormonally unresponsive, highly malignant state. ’ For patients with breast cancer, durable remissions may be achieved by treatment with cytotoxic drugs or hormonal agents, either singly or in combination. Unfortunately, although initial response rates to these agents are usually high, most patients even-. tually relapse. In the setting of relapse, breast cancers rapidly develop resistance to chemotherapy. Recent studies have identified a number of factors that may be involved in the development of clinical resistance. The multidrug-resistance phenotype To develop a model to study mechanisms of resistance 10 antineoplastic drurq in vitro, a number of laboratories have isolated drugresistant cell lines. Such lines are selected for resistance to single agents yet frequently display cross-resistance to a number of drugs that are dissimilar in both structure and proposed mechanism of action. Drugs associated with this multidrug-resistance phenotype include the anthracyclines, epipodophyllotoxins, vinca alkaloids and actinomycin D (Refs I and 2). Multidrug-resistant cells usually display decreased drug accumulation> , which is associated with the OverexFression of a surface glycoprotein known as P170, gp140-180, P-glycoprotein or
the mdr gene product5f6. The putative role of this protein as a drug efflux pump has recently been reviewed6. While overexpression of P170 is a common feature of multidrugresistant cell lines, this alteration may not complete!y account for the expression of multidrug resistance. Each multidrug-resistant cell line exhibits a unique pattern of cross-resistance. Although mutations in the mdr gene7 or other factors may alter the P170 protein it is unclear whether the diversity in resistance patterns can be accounted for at the level of the P170 protein. Defects in drug accumulation occur in most multidrug-resistant cell lines, tut the magnitude of these defects is often insufficient to account fclr the overall degree of drug :esiQance”. In addition, cell lines that display multidrug resistance without overexpression of P170 have been described’. Identifying biochemical
tumors express relatively high levels of P170, there is as yet no clear association between the development of clinical drug resistance and the expression of P170 in human tumors (Ref. 15 and J. A. Moscow et al., unpublished).
Biochemical changes in multidrug-resistant breast cancer cells To investigate the mechanisms associated with the development of multidrug resistance in breast cancer, our laboratory has selected an adriamycin-resistant MCF-7 human breast cancer c-l! !inc (MCF-71AdrR) which displays the multidrug-resistance 4 13.16
‘in_: I
r;llstance
pheno-
in MCF-7i
2:; ‘is ass#Jciated with a two- to three-fold decrease in drug accumulation. and a 45-fold increase in the expression of P170.
Detailed
analysis
revealed
that
MCF-7!AdrR cells have altemtions in the levels of a number ol: proteins’0,‘2-‘4*‘6 including several drug-metabolizing mzymes’3*‘7*‘” Drug-metabolizing enzymes are generally classified into phase I and phase II enzymes. Philse I .. enzymes oxialzc a wide rclrq!:: uf planar lipophilic toxins. For many carcinogens and procarcinogens (e.g. benzolalpyrene) the inter-
mediates
genevated
by
these
TABLE I. Properties of multidrug-resistant MCF-7/AdrA cells, xenobictic-resistant rat :Ipperplastic nodules, and estrogen receptor-negative breast cancer cells Cell type
Characterlstlc
L ER t EGFR f Tumorigenicity r Protein kinase C t GST-x i AHH Resistance to benzo[a]pyrene T P170
yes4
rat liver hyperplastic nodules
ER-negative breast cancer cell lines
z-negative breast cs?cczr?
ND ND :;a0 yes30 yes30 ye.9
yes ye? yesa yesz9 yesyesz’ yeszB
yes ye? NA ND vesz7 fiD ND
yes18
ND
ND
ND, not determined: NA, not applicable: ER, estrogen receptor; EGFR, EFG receptor.
TOXIN-SENSITIVE CELL
TOXIN-RESISTANT CELL
Fig. f Mxid of toxin resistance in breast cancercelfs.f. toxin; T-OH,oxygenafed foxin; TX, foxin wnjugaf@: P170, toxin efffux pimp; ER, estrogen receptor; EGFR, epkkrmalgrowfhfactctrrecepbr.Intoxin-sensitive cells (lefi) acfivity ofphasef drug-melabolizingenzymes (1)is high,leading ic7high concenfratims of thrr oxygenaled toxin producfs, which bind DNA 13); the level of estrogen ~eceprors is afsr, high. In fu~#-fesfsfa~t &Es (righ!) activityofpmst3 I dr~~-~~~ab~[~~~~ t%ZWk?s(I) iS#Ck.Ke~and t&at&p&Se HenqJRes is i#crt?ased,fr?i&ir?g tc reduce&accumula&%t and cyrfmxicity (3) of oxygenated foxin; PI 7%mediated &f/m (4) wd EGF receptor d&?&y are great& irnxeased. The roles of fbe estrogen and EGF receptors in toxic.iry are unclear.
are, paradcxicaliy, more binders ot DNA, 2Ed are more cytotoxic 17. However, the products of the phase f reactions
enzymes potent
arc also sub;s:r;ri~s of phase II drugmetabolizing enzymes: these enzymes conjugate the products of the phase I reactions with hydrophilic moieties such as glutathione or glucuronide. The resulting conjugates are readily excreted from the cell. In MCF-71AdV”cells, the activity of the anionic isozyme of the phase 11 enzyme glut Ithione-S-transkrase (GST-n) is increased 45fold’“*“. (11 addition, m~ltidrug resistance in MCF-7/A&” is associated with downregulation of the phase I enzyme arylhydrocarbon hydroxylase (AHH)‘“. The roles of GST and AHH in resistance to drugs associated with multidrug r&stance are currently under investigation; a possible mechanism is shown in Fig. 1. The alterations in drug-rn~tabo~~~ing enzymes and other proteins in MCF7lAdr” emphasize that the development of multidrug resistance in breast cancer may be associated with multiple biochemical changes.
Multidrugresistame and carcinogen resistance The biochemical ajteratians associated with wkuttidrug wsistanrp
in MCF-7/Adp, including increased expression of P170 (Ref. 4) and GST-n (Refs 23 and 16) and downreguiatian of AHH’” bear a striking similarity to alterations observed in rat premalignant hyperplastic liver nodules in the Solt-Faber model of chemical carcinogencsis16**g. Although these nodules are induced by a variety of carcinogens, they resemble multidrug-resistant cells in that they develop resistance not only to the agent used in their selection, but also to a wide range of hepatatoxins. indeed, studies indicate that rat hyper~lasti~ nodules develop resistance to a wide range of antineoplastic agen tszol while our laboratory has shown that multidrug resistance in MCF7/AdvR is associated with CTQSSresistance to the carcinogen ~en~~~~~py~ene~~.
The similarities between rat hyperplastic nodules and MCF-7! 24d2”cells raise the possibility that tumors derived from tissues with exposure to chemicalbcarcinogens may be predisposed to be resistant to anticancer agents. Clinirat evidence appears to supporl this, as tumors of the colon and lung tissues with exposure to relatively high levels of carcinogens - are USUdl~ refractory to chemotherapy in contrast, tumors derived from tissues with lOl%F Ievzit:
af exposure
to carcinogens (such as lymphomas) appear to acquire the resistance phenotype only after exposure to chemotherapy. Cross-resistance to antiestrrrgens As breast cancer may be swccessfully treated both by cytotoxic drugs and hormonal agents, we were interested in whether the development of multidrug-resistance in MCF-7/AArR resulted in any alteration in the hormonal sensitivity of the cell line. Wildtype MCF-7 cells are an excellent model system for the study of hormone-res~~nsi~~e breast cancer, as they contain receptors for both estrogen and progesterone, and are recponsive to physiological concentrations of estrogcns2’. The development of multidrug resistance in the MCF-Y’/Anr”R cell line was found to be associated with a loss of response to estrogen and cross-resistance to the antiestrogen 4-hydro~ytamo~~f~n2~” Furthermore, while wild-type MCF-7 cells require estrogen to form tumors in athymic mice, the alterations in hormonal sensitivity in MCF-71AdP have resulted in the cells being able to form tumors equally well in the absence or presence of estrogenzz. The development of hormonal ~~~en5it~vity in MCF-?/A&” is associated with loss of theestrogen
TlPS - December 2988 [Vol. 91 receptor. In contrast, this cell line displays a 100-fold increase in levels of the epidermal growth factor (EGF) receptor, and a loss of mitogenic response to EGF (Ref. 22). Other multidrug-resistant cell lines have been reported to have increased levels of EGF receptos3, although the magnitude of the change appears to be larger in MCF-7/A& R. Previous studies have shown that acute treatment of cells with cytotoxic drugs can decrease levels of the estrogen receptos4 and increase levels of the EGF recepto?‘. Although associated with cytotoxicity, these effects were rapidly reversible upon removal of drug from the medium. It remains unclear whether the mechanisms responsible for these transient alterations in receptor levels bear any relationship to the stable recaptor alterations induced in MCF7’iAdp following chronic drug treatment. A common mechanism of resistance to toxins? The finding that the development: of multidrug resistance in MCF-7/A&” is associated with multiple biochemical changes, including alterations in the expression of AHH and GST-x, as well as the loss of estrogen receptors, prompted us to examine the expression of these drug-metabolizing enzyme8 in other breast cancer cell lines with various levels of estrogen receptors. These studies revealed that the estrogen receptor-positive cell lines possessed significantly higher levels of AHH activity than the estrogen receptor-negative cell line++‘. Other studies from our laboratory have shown an inverse relationship between GST-n: activity and estrogen receptor levelsz7. These alterations are associated with resistance to both ellipticine and benzofalpyrene in estrogen receptor-negative cell line?. Protein kinase C activity has also recently been shown to bear a relationshi p to both multidrug resistance’ e2s and the estrogen rece tor status of breast cancer cells Pg. As previously described, the development ot resistance to carcinogens in rat hepatocytes and cytotoxic drugs in MCF-7/A@ is associated with a similar pattern of biochemical alterations. Our findings suggest that a number of these
445 alterations are also related to levels of estrogen receptor in breast cancer cells (Table I and Fig. 1). In addition to providing an invitro model of multidrug resistance in breast cancer, the development of hormonal insensitivity in MCF7/Adra bears analogies to the clinical progression of breast cancer. It is well established that estrogen receptor- and progesterone receptor-negative tumors have a poorer prognosis than those possessing these receptors. Furthermore, increased levels of the EGF receptor in primary breast cancer have been reported to be associated with tumors having the poorest profinosis32,33. Selection of MCF-7/A&” has therefore resulted in the progression from estrogen receptorand progesterone receplor-positive, hormone-responsive breast cancer cells to cells displaying features of the poorest prognosis breast tumors: estrogen receptorand progesterone receptor-negative, increased levels of the EGF receptor, resistance to drugs and hormonal agents, and increased tumorigenicity. q
q
0
biochemical The multiple changes associated with multidrug resistance in MCF-7/A&” define a conserved pleiotropic response to cytotoxins, and appear to be relevant to the progression of breast cancer in viva. MCF-71AdrR cells therefore represent a useful model to study both the mechanism of multidrug resistance in breast cancer and the transition of breast cancer to a hormonally unresponsive, highly malignant state. The finding that a similar pattern of gene expression occurs in a multidrug-resistant MCF-7 breast cancer cell line, rat hyperpiastic nodules and estrogen receptornegative cells suggests that similar factors regulate biochemical alterations associated with these three model systems. It is a major challenge to determine both what regulates these alterations and the role that each of the biochemical changes may play in resistance to antineoplastic drugs, chemical carcinogens and hormonal agents. References 1 Vtckers,
P. J.,
Townsend, A. 1. and
Cowan, K. H. CRC Crib Rw. Dev. Cffttcer Cttcrtrothrr
(in $-TSS?
2 Moscow, J. A. and Cowan, K. H. (1988) J. h’uff Cancer Inst. 80, 14-20 3 Skovsgaard, T. (1978) Cancer Reg. 38, 4722-4727 4 Fairchild, C. R. et al. (1987) Cancer Res. 47, 5141-5148 5 Riordan, J. R. and Ling, V. (1985) P~ffr~scol. 7&r. 28.51-75 6 Cottesman, M. M. and Pastan, I. (1988) Tresds Pharmacol.
Sci. 9, 54-58
7 Choi, K.. Chen, C-j., Kriegler, M. and Roninson, I. B. (1988) Cell 53,519-529 8 Sirotnak, F. M., Yang, C. H., Mines, L. S., Oribe, E. and Biedler, J. L. (1986) J. Ceil. Physiol. 126, 266-274 9 McGrath,.T. and Center, M. S. (1987) Biochem. Biophys. Res. Commun. 145, 1171-1176 10 Fine, R. L., Pate!, J, and Chabner, B. A. (1988) Droc. Nat1 Acnd. Sci. USA 85, 582-586 11 van der Blieck, A. M., Meyers, M. B., Riedler, J. L., Hes, E. and Borst, P. (1986) EM80 J. 5,3201-3208 12 Yeh. G. C.. Occhiointi. S. ‘1.. Cowan. K. I%, Chadner, B. x. and b&&s, C. E: (1987) Cancer Res. 47.5994-5999 13 Batist, G., Tulpule, A., Sinha, B. K., Katki, A. G., Myers, C. E. and Cowan, K. H. (1986) I. &%I. Chem. 261, 1x%4-15549 14 Kramer, R. A., Zahker, J. and Kim, J. (1988) Science 241, 694-697 15 Fojo, A. T., Ueda, K., Slamon, D. J,, Poplack. D. G., Gottesman, M. M. and Pastan, f. (1987) Proc. Natf Acad. Sci. USA 84,265-269
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