[CANCER RESEARCH 55, 5063-5068, November 1, 1995] Characterization of A and B Expression in Human Breast Cancer1

J. Dinny Graham,2 Christine Yeates, Rosemary L. Balleine, Suzanna S. Harvey,3 Jane S. Milliken, A. Michael Bilous, and Christine L. Clarke Department of Medical Oncology, University of Sydney ¡J.D. G., C. Y., R. L B., S. S. H., C. L. C./, and Department of Anatomical Pathology. ¡J.S. M.. A. M. fi./. Westmead Hospital, Wi'Mmead, MMVSouth Wales 2145, Australia

ABSTRACT binding to c/.c-acting progestin-responsive elements on DNA and modulation of the activities of target genes (2, 3). PR is detected in the The human (PR) is a ligand-activated nuclear chick and human as two distinct proteins of different molecular that mediates progesterone action in target tissues. weights (4, 5); in humans the PR-A protein is M, 81,000-83,000 and Two PR proteins, PR-A (Mr 81,000-83,000) and PR-B (Mr 116,000- PR-B is M, 115,000-120,000 in size (6-9). The two proteins, which 120,000), have been described and different physiological activities as differ only in that PR-A lacks the first 164 amino acids contained in cribed to each on the basis of in vitro studies, suggesting that their ratio of expression may control progesterone responsiveness in target cells. Pres PR-B, are translated from distinct mRNA subgroups transcribed from ence of PR in breast tumors is an important indicator of likely respon a single gene under the control of separate A and B promoters (10). siveness to endocrine agents. However, the relative expression of PR-A Although both PR-A and PR-B bind progestins and interact with and B in breast cancer has not been described, and its clinical significance progestin-responsive elements, there is increasing evidence that they has not been addressed. Expression of PR-A and B was measured by are functionally different. In transfection studies, the two proteins immunoblot analysis of 202 PR-positive human breast tumor cytosols. The have different abilities to activate progestin-responsive promoters, and ratio of expression of the two PR proteins (PR-A/B) ranged from 0.04 to these differences are promoter and cell specific (11-14), suggesting 179.3. The median PR-A/B ratio was 1.26, and 61.4% of samples had PR-A/B ratios between 0 and 2. PR-A/B ratios deviated significantly from that cellular responsiveness to progestins may be modulated via a normal log distribution; tumors containing a PR-A/B ratio greater than alterations in the ratio of PR-A and B expression. Whereas PR-B tends 4 were overrepresented in the group. Linear regression analysis revealed to be a stronger activator of target genes in many settings, PR-A has that high PR-A/B ratios, in general, derived from a low concentration of been shown to act as a dominant repressor of PR-B (13, 14), suggest PR-B rather than high expression of PR-A. PR-A/B protein ratios were ing that high PR-A expression may result in reduced progestin re not correlated with the age of the patient or with total PR concentration. sponsiveness. Furthermore, PR-A has been shown to diminish the A third PR protein band (PR78kDa) was detected in a number of samples response of other hormone receptors such as the androgen, glucocor- and comprised greater than 20% of total PR protein in 52 (25.7%) of the ticoid, mineralocorticoid, and estrogen receptors to their cognate 202 tumor samples examined. The range or frequency distribution of ligands (15-17). It has also been reported that PR-A but not B can PR-A/B ratios in samples containing PR78kDa was not different to the overall group. In summary, in PR-positive breast tumors, the ratio of confer antiestrogenic actions to progestin antagonists (16), possibly expression of PR-A and B proteins is close to unity, as is seen in a number via noncompetitive binding of PR-A to specific transcription factors of other progestin target tissues. However, a significant proportion of required for ER and PR action. tumors expressed very low levels of PR-B and a consequently high PR-A/B Approximately equimolar expression of PR-A and B is observed in ratio. Although the clinical consequence of this observation is not known, chick oviduct (4) and human uterus (6), and a similar ratio of expres the in vitro findings that PR-A may act as a represser of PR-B suggest that sion is seen in human breast cancer cells in culture (8). In contrast, PR tumors containing primarily PR-A may identify a subset of patients with in the rabbit uterus is expressed as a single form homologous to low or aberrant response to endocrine agents. human PR-B (18). In the rodent, PR-A expression predominates over PR-B in a 3:1 ratio (19, 20). The biological importance of these INTRODUCTION different ratios of PR expression has not been explored extensively. The PR4 is a specific and effective mediator of progestin action in Little is known of whether relative PR-A and B expression is modu target tissues such as the breast and endometrium and is essential in lated in vivo, except in the chick oviduct, where alterations in the ratio the complex control of proliferation and differentiation at various of PR forms during late winter or in aged, nonlaying animals results stages of development (1). PR is a member of a superfamily of in a measurable decrease in PR functional activity (21, 22). ligand-activated nuclear transcription factors and is composed of The functional differences between PR-A and B that have been specific domains involved in DNA and hormone binding and trans- described in vitro imply that response to progesterone may be mod activation (2). Progestin activation of PR in target tissues is mediated ulated by differential expression of the two PR forms. Clearly, if these via dimerization and phosphorylation of the receptor, resulting in observations are borne out in vivo, the balance of expression of the two receptor forms may be critical in the control of progesterone Received 5/1/95; accepted 8/28/95. responsiveness in normal and malignant tissue. PR is an important The costs of publication of this article were defrayed in part by the payment of page marker of responsiveness in breast cancer, and the presence of PR in charges. This article must therefore be hereby marked advertisement in accordance with ER-positive breast tumors indicates likely responsiveness to endo 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by grants from the National Health and Medical Research Council crine agents (23, 24). Absence of PR is associated with markedly (NHMRC) of Australia, the University of Sydney Cancer Research Fund, and the New reduced response to endocrine agents (25). Given that the two PR South Wales Cancer Council. J. D. G. is supported by an NHMRC Dora Lush Biomedicai proteins may be functionally different, the ratio of expression of PR-A Scholarship and by the Westmead Hospital Research Institute. R. L. B. is supported by an NHMRC Medical Scholarship. and B may be as important as the absolute PR level in determining 2 To whom requests for reprints should be addressed. Phone: 61-2-6336954; Fax: endocrine response. The relative expression and regulation of PR-A 61-2-8916035; E-mail: dinny(

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1995 American Association for Cancer Research. PROGESTERONE RECEPTOR IN HUMAN BREAST CANCER MATERIALS AND METHODS 12345678 Materials. Chemicals used were of analytical reagent grade and were obtained from the sources listed previously (26). The anti-PR mAbs, hPRao and hPRa7, which have been described previously (27), were concentrated by ammonium sulfate precipitation of hybridoma supernatants and dialysis in PBS PRB* containing 0.02% sodium azide. The dilutions used for saturating detection of PR on immunoblots were determined for each batch. Abbott PgR-EIA mono clonal and ER-EIA monoclonal reagents for EIAs were from Abbott Labora tories (Diagnostics Division). Preparation of Tumor Cytosols. Biopsy samples were collected at the time of breast lump excision and frozen at -70°C. The tissues were pulverized in a precooled vessel, then homogenized at 4°Cin a buffer containing 10 mM Tris pH 7.4-7.5, 1.5 mM EDTA, 5 mM sodium molybdate, and 1 mM mono- thioglycerol. Homogenates were centrifuged at 436,000 x g for 20 min at 4°C, and then cytosols were transferred to fresh tubes. Protein concentrations were determined by a method based on that of Lowry et al. (28), as recommended in notes accompanying the Abbott EIA reagents. PR and ER concentrations were determined by EIA. The cytosols were stored up to 18 months at —¿80°C. PR A/B v V Protein Electrophoresis and Immunoblot Analysis. Tumor cytosol pro teins (300 fig/lane) were separated by electrophoresis through 7.5% polyacryl- Biof amidc gels containing SDS and were electroblotted overnight onto nitrocellu vj00*£>2 lose as described previously (26). PR proteins were detected using anti-PR mAbs; hPRa7 recognizes both PR-A and PR-B proteins, and hPRao detects tumorsO\ PR-B protein but not PR-A (27). Both hPRao and hPRa? were used in combination (each at 1:1000) for detection of tumor PR proteins. Specific PR protein bands were visualized on autoradiographic film using a horseradish peroxidase-conjugated goat antimouse secondary antibody (Dako, Australia) at a 1:5000 dilution and enhanced chemiluminescence detection substrates (Am- ersham, Australia). A cytosol made from the T-47D breast cancer cell line (29), 5jj which is highly PR positive, was included on each gel as a positive control. 4¡ Band intensities were measured densitometrically using a Molecular Dynamics 31 densilometer and Imagequant software. The linear range of detection of PR on immunoblot was established by a standard curve constructed using increasing concentrations of T-47D cytosol PR. This was analyzed by densitometry and established the relationship between PR concentration and linearity of detec tion by this method. Multiple exposures were always made from each immu 0•ililn1LiiL»4-3-2-10123456Ini noblot of tumor cytosols, and results were taken only from those that fell within this linear range. More than one exposure of an immunoblot was generally analyzed, and the results were compared and found to be close to PR A/B identical in most cases. Results that were outside the linear range were omitted Fig. 1. Immunoblot analysis of PR in breast Iunior cytosols. A, cytosols were prepared from the analysis. PR-A/B ratios were calculated by dividing PR-A AU by from PR-positive primary breast tumor biopsies as described in "Materials and Methods." PR-B AU. In a small number of samples (n = 6) where PR-A or B expression Each sample (300 fig; Lanes 1-7) was run on a 7.5% polyacrylamide-SDS gel with was visible by eye, but so low as to be abolished by the background correction T-47D-positive control cytosol (15 /ig; Lane 8) and transferred to nitrocellulose. PR protocol of the densitometry software, this correction was switched off to allow proteins were visualized using hPRao and hPRa7 antibodies and chemiluminescent detection. Bands representing PR-A, PR-B and PR78kDa proteins are indicated. The estimation of the PR-A/B ratio. However, for consistency with the total sample PR-A/B ratio in each sample was calculated by dividing PR-A AU by PR-B AU. The population, the correction for background in these samples was taken into PR-B levels in Lanes 5-7, although extremely low. were measurable but are not visible ¡n account when calculating absolute expression levels (see below). this exposure. B, frequency distribution of PR-A/B protein ratios. The PR-A/B protein To allow correlation of PR concentration with other parameters, such as PR ratios determined in breast tumors, as described above, were natural log transformed and expressed as a frequency distribution (n = 202). and ER EIA and age, tumor PR protein levels were normalized to total PR concentration in the T-47D-positive control cytosol. This was done by dividing the total immunoreactive PR protein AU in the tumor sample by the total ¡mmunoreactive PR AU in the T-47D cytosol from the same immunoblot. The migrated slightly ahead of the M, 81,000-83,000 PR-A band seen in normalized PR values represent the amount of PR per 300 jxg cytosol protein the positive control breast cancer cell line T-47D (Fig. \A, Lane 8) as a percentage of PR in 10 /j,g T-47D cytosol protein. Linear regression and (Ref. 8). This difference in migration is likely to be due to the Spearman's rank correlation analyses were carried out using SPIDA and difference in protein loading rather than representing a significant Statview (Abacus Concepts, Inc.) statistical analysis software. difference in size between tumor and T-47D cell PR-A. Alternatively, the difference in migration may reflect a difference in the level or number of sites of phosphorylation. A third immunoreactive band RESULTS (PR78kDa) migrating ahead of PR-A at approximately M, 78,000 was Cytosols from 202 PR-positive primary tumors were analyzed by detected in a proportion of tumors (Fig. IA, Lanes 2, 6, and 7). Some protein immunoblot. Ninety % (182) of these tumors were also ER faint, low molecular weight, immunoreactive bands were seen occa positive by EIA. Fig. 1 shows a representative immunoblot of tumor sionally in individual cytosols (see Fig. IA, Lanes 5-7, for example). cytosols probed for PR. The PR-B protein band was visualized as a However, these bands had different mobilities in different samples group of two or three closely migrating bands with a mobility of Mr and were seen in only a small proportion of cases. 115,000-120,000, as reported previously in other tissues (6, 8, 9, 27, All tumor samples used in the study were PR positive by both 30). An immunoreactive band representing PR-A protein was also immunoblot and EIA and represented 78% of the total number that detected in most tumor cytosols (Fig. \A, Lanes 1-7). This band were PR positive by EIA. To correlate levels of PR detected on 5064

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1995 American Association for Cancer Research. PROGESTERONE RECEPTOR IN HUMAN BREAST CANCER FRA rank correlation analysis (Fig. 4). This negative correlation between PR-B concentration and A/B ratio was nonlinear at low levels of PR-B. which suggests that high PR-A/B ratios were explained by low levels of PR-B expression rather than high PR-A. This relationship PRB between PR-A and B protein expression is described by the fitted model calculated from the linear regression line of best fit; PR-A concentration = eÃŒA3ìX(PR-B concentration)"•55<<(shownin Fig. 4, -2 O 2 4 6 8 10 inset). In PR concentration (AU) A faster migrating M, 78,000 PR protein was detected in 52 of 202 Fig. 2. Expression of PR proteins in breast tumors. Immunoreactive PR-A and PR-B in (25.7%) tumors at levels greater than 20% of total PR concentration breast tumor cytosols were quantitated densitometrically from immunoblots and normal (Fig. 5). This band was detected only by the hPRa7 antibody, which ized to total PR in concurrently run T-47D-positive control cytosol to correct for loading. detects both PR-A and B, and not by hPRao, which recognizes PR-B This was done by dividing PR-A AU in tumors by total PR protein AU in T-47D and PR-B AU in tumors by total PR protein AU in T-47D. O and bars, median value and alone (data not shown), suggesting that this protein may represent an interquartile range, respectively, representing AU (n = 140). NH2 terminally truncated form of PR. The distribution of PR-A/B

Table I Distribution of PR A/B ratios in breast tumors" A IO] PR-A/Bratio0-11-22-33-40-44-5tí£4No.oftumtirsSK361612152143650Percentoftotal43.617.87.95.975.26.917.924.8 S 8

PU 6

" PR A/B ratios were calculated by dividing PR A AU by PR B AU. The median PR l 41 A/B ratio was 1.26, minimum = 0.()4, and maximum = 179.3. The numbers of tumors with A/B ratios in the indicated ranges are shown. ~ 2' •¿**%.••*,. immunoblots with EIA values, concentrations of immunoreactive PR were quantitated in arbitrary units and normalized against PR protein expression in the positive control T-47D. Normalized immunoreactive -4 -3 -2 -1 0123 456 PR level was correlated with level of PR measured by EIA In PR A/B (P = 0.0001; data not shown). PR-A and B protein levels are shown in Fig. 2 with median values and interquartile range indicated. The range of expression of PR-A and B was variable, although the median B 6 and interquartile ranges for both proteins were similar, reflecting the fact that most tumors contained both proteins in similar proportions (see below). Relative expression of PR-A and B proteins was calculated in all samples and expressed as a frequency distribution of natural log- transformed PR-A/B ratios (Fig. Iß).Statistical analysis, using a co Shapiro-Wilks test for normality, of the distribution of the natural log-transformed PR-A/B ratios revealed that the values did not fall within a normal log distribution (P = 0.023) due to an overrepresen- ftH 0 tation of tumors with a high PR-A/B ratio. This is evident in the frequency distribution (Fig. IB), which is asymmetrical due to the cohort of tumors with very high PR-A/B ratios. The distribution of -2 PR-A/B ratios is shown in Table 1. There was no correlation between the overall level of PR expression in tumor cytosols and PR-A/B ratio (Fig. 3A). The ratio of PR protein expression was not related to age (Fig. 3fl), suggesting that the PR-A/B ratio is unlikely to be related to -4 menopausal status. O 20 40 60 80 100 PR-A/B ratios ranged between 0.04 and 179.3, with a median ratio of 1.26 and an interquartile range of 0.57-3.88. The majority of age (years) tumors (61.4%) had a PR-A/B ratio between 0 and 2, and 75.2% of Fig. 3. Correlation of PR-A/B ratios with age and total PR protein level. A. PR-A and tumors had a PR-A/B ratio between 0 and 4 (Table 1). Tumors with B proteins in breast tumor samples were quantitated densitometrically from immunoblots, PR-A/B ratios greater than 4, above the upper quartile range of 3.88, and the PR-A/B ratio in each sample was calculated by dividing PR-A AU by PR-B AU. The values were natural log transformed and correlated to In (PR concentration) normal comprised 24.8% of the total (Table 1). Immunoblots revealed that ized to total PR in T-47D cells (n = 140). Tumor PR protein levels were normalized to these tumors generally contained low levels of PR-B (see Fig. 1/4, total PR concentration in the T-47D-positive control cytosol by dividing the total immu Lanes 4-7, for example), and this was borne out by statistical anal noreactive PR protein AU in the tumor sample by the total immunoreactive PR-AU in the T-47D cytosol from the same immunohlot. The normalized PR values represent the ysis. There was a negative correlation between PR-A/B ratio and amount of PR per 300 ¿igcytosol protein as a percentage of PR in 10 fig T-47D cytosol PR-B concentration, which was statistically significant by Spearman's protein. B. In (PR-AB ratio) was correlated to the age of the patient (n = 181).

5065

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1995 American Association for Cancer Research. PROGESTERONE RECEPTOR IN HUMAN BREAST CANCER ratios in samples containing PR78kDa, which had a median value of 12 3456 1.93, did not differ significantly from that of the entire tumor cytosol group. Tumors containing PR78kDa did not represent a unique sub group of the overall population in regard to the other parameters examined, since expression of the protein was not correlated with total PR concentration (Fig. 5ß)or PR-A/B ratio (P = 0.76), and no PRB relationship was seen between the presence of PR78kDa and age (not shown). PRÄ. PR78kD DISCUSSION

The relative expression of PR proteins has not been examined previously in human breast tumors. Expression of PR-A and B was PR78kD 9 15 54 55 85 91 measured on immunoblots of PR-positive primary breast tumor cy- (%total FR) tosols. The median PR-A/B ratio in a group of 202 breast tumors was close to unity. This result agrees with the equal expression of PR-A and B observed in chick oviduct (4) and human uterus (6), and a similar ratio of expression is seen in human breast cancer cells in R »3"PHr— culture (8). In contrast, PR in the rabbit uterus is expressed as a single •¿â€¢*• form homologous to human PR-B (18). In the rodent, PR-A expres 1«3•4-1OÖ sion predominates over PR-B in a 3:1 ratio (19, 20). These different tissues have differing functional requirements for PR in terms of progestin responsiveness. Possibly differential PR-A and B expression is one mechanism by which their distinct physiological requirements vuIHV3mQ00tse^100-'80-,60-140-.20-•* are fulfilled by this relatively well conserved but differently expressed 0.•':? protein. Indeed, even in a particular tissue type such as the breast, control over relative PR-A and B expression may be important in "• modulating progestin responsiveness as required at different times in the normal ovarian cycle. *~ V¿"• Whether the ratio of PR-A and B proteins in target cells is fixed or 0¿lÕklLÕwi"% ji •¿ whether it can be altered under physiological conditions is poorly understood. In mammalian target tissues PR expression is under dual * * •¿*•" control by estrogen and progesterone (24, 31), with their coordinate regulation of PR concentration resulting in modulation of responsive 02468 10 ness to progestins. In the normal human endometrium and in breast In (total PR) cancer cells, progestin exposure leads to a similar decrease in the Fig. 5. Expression of PR78kDa in breast tumor cytosols. A, cylosol proteins from PR-positive primary breast tumor biopsies were analyzed by ¡mmunohlot and ehemilu- minescent detection. Lanes 1-6 contain different tumor cytosols loaded at 3(H) ¿¿g/lane. 100- Bands representing PR-A. PR-B. and PR78kDa proteins are indicated. The relative 6 amount of PR7SkDa in each sample is indicated below each lane, expressed as a percentage of total PR expression (AU), calculated as the sum of PR-A. PR-B, and PR78kDa in that sample, ß,correlation of relative PR78kDa expression with total PR 41 concentration calculated by normalization of total PR AU in the tumor cytosol to total PR AU in the T-47D control (n = 140). pa

concentration of both PR-A and B (5, 6) resulting in no change to PR-A/B. Retinoic acid, which decreases PR mRNA and protein levels O' in breast cancer cells, resulting in diminished cellular responsiveness to progestins, is equally effective on both PR-A and B (26, 32). -2- However, there is some evidence that estrogen, which is the major physiological modulator of PR levels in vivo, can alter the relative expression of PR-A and B. Estrogen induces PR mRNA and protein -4 in most target cells (20, 33-37). In breast cancer cells this effect is -4-20 24 6 io mediated primarily through PR-B, resulting in a decrease in PR-A/B In PR B (AU) with increased PR expression.5 In the rat uterus (where PR-A pre dominates), estrogen withdrawal results in a decrease in PR-A/B from Fig. 4. Regression analysis of PR-A/B ratio with PR-B concentration. PR-A and B concentrations in breast tumor samples were determined from immunoblots. The PR-A/B 3.2 to 1.9 (19). ratio in each sample was calculated by dividing PR-A AU by PR-B AU. PR-B concen Although the median PR-A/B ratio in the breast tumor cytosols was tration was calculated by correcting PR-B AU for loading by normalizing to total PR AU close to 1, a number of tumors had PR-A/B ratios that differed quite in concurrently run T-47D cytosol. PR-B and PR-A/B were natural log transformed and compared by linear regression analysis. The data are shown {•)against the line of fit dramatically from this value, resulting in a nonnormal distribution of determined by linear regression (O; n —¿137).Inset, the line of fit equation [PR-A PR-A/B. In particular, samples with high PR-A/B ratios were over- concentration = e1-431 X (PR-B concentration)"556] for the natural log-transformed data, determined by linear regression, was used to describe the relationship between A and B protein. PR-A AU concentration was plotted against PR-B AU as predicted by the fitted SJ. D. Graham, S. D. Roman, E. McGowan, and C. L. Clarke, unpublished equation. observations. 5066

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1995 American Association for Cancer Research. PROGESTERONE RECEPTOR IN HUMAN BREAST CANCER represented in the tumor population. Analysis by Spearman's rank regulated promoters give rise to distinct mRNA species that are correlation revealed a significant negative relationship between PR-B differentially expressed in different cell lines and tissues (42). A expression and PR-A/B. Further linear regression analysis of the second NH2 terminally truncated human AR protein has been de natural log-transformed data showed that, whereas the inverse scribed, and the two AR isoforms have been postulated to be func tionship observed between In PR-A/B and In PR-B remained linear for tionally different (43). Furthermore, the suggestion that the transcrip most values, this relationship was lost at low levels of PR-B. Further tional activity of PR-B may be negatively regulated by PR-A is not more, it was these samples that gave rise to the high PR-A/B ratios, unprecedented. A number of reports have suggested that an activator which were overrepresented in the tumor population. and repressor can be encoded by the same gene (44). The transcrip The consequences of high PR-A/B ratios on progestin responsive tional regulation of the TR gene by its isoforms is a case in point. ness in human tissues are not known. However, there is increasing Three TR isoforms (al, a2, and ß)are generated as the result of evidence that PR-A and B are functionally different. In the chick alternative splicing. The a2 form is transcriptionally inactive alone oviduct, relative expression of PR-A and B is under seasonal and but blocks activation by the al or ßforms of TR, even when these developmental control. Alterations in the ratio between the PR forms activators are in 2-fold molar excess (45). The murine transcription during late winter or in aged, nonlaying animals results in a measur activator, mTFE3, which binds to the intronic enhancer of the immu- able decrease in PR functional activity (21, 22). Although both PR noglobulin heavy chain gene, acts in a similar manner. A natural proteins are able to mediate progestin-activated transcription in trans- splicing variant. mTFE3-S, acts as a dominant negative repressor of fection studies, they differ in their efficacy and specificity for target mTFE3 activation in a highly ratio-dependent manner (46). genes. Whereas PR-B is able to activate mouse mammary tumor A significant proportion of the tumor samples examined contained virus-chloramphenicol acetyltransferase more effectively (12), PR-A a M, 78,000 PR protein. The presence of PR78kDa was not correlated is known to stimulate a tyrosine aminotransferase progestin-respon- with PR-A/B ratio, and the source of this PR species is not yet clear. sive reporter to a greater extent than is PR-B (14). PR-A also acts The band was not detected by hPRao, which recognizes only PR-B, synergistically with estradiol to induce the ovalbumin promoter, suggesting that it may represent an NH2 terminally truncated form of whereas PR-B is inhibitory under the same conditions (11). Further the receptor. There are no possible sites of alternative initiation of more, there is emerging evidence that, whereas PR-B is, in most translation contained in the NH2-terminal region of the PR gene,which contexts, a transcriptional activator, the ability of PR-A to function as would predict a protein of this size. A methionine at amino acid an activator is more restricted; in settings where both forms activate position 595 has been suggested as a possible site of alternative transcription, PR-B is more effective than PR-A (13, 17, 38). The initiation, and this would give rise to a M, 45,000-50,000 protein observed differences in PR-A and B activity have focused attention on termed PR-C (47). However, no immunoreactive band of that size was the NH2-terminal region of PR, which is unique to PR-B. PR mediates detected in the tumor cytosols examined. It is possible that the progestin regulation through specific AF domains contained in the presence of PR78kDa reflects expression of a PR variant. There is amino-terminal (API) and ligand-binding (AF2) domains (39). It has evidence from breast tumor mRNA studies that several ER variants been postulated that the PR-B-unique NH2-terminal portion of PR may exist (25, 48). In particular these comprise whole exon deletions binds an intermediary factor involved in activation by these domains ascribed to splicing errors. A number of the exons of PR are between (12) or is, itself, a third activation function domain (AF3), and that this 100 and 150 bp in length (49). so deletion of any of these could accounts for the functional differences between PR-A and B (40). potentially predict a size difference in the order of M, 5000-6000, as PR-A has been shown to act as a dominant repressor of PR-B function. seen between PR78kDa and PR-A, allowing speculation that this Cotransfection of increasing amounts of PR-A with PR-B resulted in protein may represent a variant PR-A. Further characterization of the suppression of PR-B activation of a progestin responsive reporter, mouse origin of PR78kDa in breast tumors is currently under way. mammary tumor virus-chloramphenicol acetyltransferase (13, 14). In the In summary, whereas the majority of PR-positive breast tumors presence of progestins or the progestin antagonist RU486, PR-A re contain similar levels of PR-A and B, a significant proportion dem presses transcriptional activation by cotransfected glucocorticoid recep onstrates low PR-B and, consequently, a high ratio of PR-A/B. Al tors, mineralocorticoid receptors, ARs, and ERs (15-17). Furthermore, though the functional consequences of unequal expression of PR-A PR-A mediates antiestrogenic effects of progestins and RU486 on en and B are not known, in vitro evidence suggests that an abnormal ratio dogenous ER in MCF-7 breast cancer cells cotransfected with a vitel- of PR expression may be indicative of an impaired or aberrant logenin ERE reporter construct ( 17). Contrary to this, PR-B and not PR-A progestin response. The implications of altering PR-A/B ratio on inhibited ER activation of pS2 and cathepsin D promoter constructs in the cellular responsiveness to progestins remain to be determined. same cell line when treated with the progestin R5020 but not RU486 (41). Clearly, the effect is complex and depends strongly on the cellular and ACKNOWLEDGMENTS promoter context. The nature and level of sensitivity of this effect in breast tumors is not known; however, if these findings are true in vivo, The authors thank Dr. Karen Byth (consultant statistician to the Division of then relative expression of PR-A and B in breast tumors may strongly Medicine. Westmead Hospital) for statistical analyses and advice. We thank influence cellular responsiveness to endocrine agents. Clearly, a tumor Dr. Wilbur Hughes and the Dcparlment of Hacmatology (Institute of Clinical with the maximum observed PR-A/B ratio of 179.3, containing almost Pathology and Medicai Research, Westmead Hospital) and the Westmead exclusively PR-A, will respond in a different way to endocrine agents Hospital Research Institute for their support. than a tumor in which PR-B predominates. If PR-B is the more tran- scriptionally active of the two PR forms, it could be anticipated that REFERENCES tumors with a high PR-A/B ratio may be the least likely to respond to 1. Clarke, C. L.. and Sutherland. R. L. Progestin regulation of cellular proliferation. Endocr. Rev., //: 266-301, 1990. endocrine agents. Work is currently under way to address whether any 2. Tsai, M-J.. and O'Mallcy. B. W. Molecular mechanisms ol action of steroid/thyroid correlation is seen between PR-A/B and other known markers of respon receptor superfamily members. Annu. Rev. Biochem.. 63: 451-486. ll)94. siveness. 3. 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J. Dinny Graham, Christine Yeates, Rosemary L. Balleine, et al.

Cancer Res 1995;55:5063-5068.

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