EXPERIMENTAL EVIDENCE FOR BIOSYNTHESIS OF STEROIDS IN METASTATIC TISSUE ORIGINATING FROM A PRIMITIVE ADRENOCORTICAL CARCINOMA
YVAN TOUITOU*, ANDRE BOGDAN and ANDRE AUZEBY Faculty of Medicine Pitié-Salpêtrière, Department of Biochemistry, 91, boulevard de l’Hôpital, 75634 Paris Cedex 13, France
(Received 6 July 1982)
Abstract-1. Cortisol, cortisone, 11-deoxycortisol and deoxycorticosterone were synthesized in large amounts in vitro by a metastatic tissue from an adrenocortical carcinoma.
2. Both 118- and 21-hydroxylase were very active.
3. A secreting metastasis can be thus responsible for a biological relapse.
4. A metastasis originating from another secreting adrenocortical carcinoma was found to be non- secreting.
INTRODUCTION
Carcinoma of the adrenal cortex is a rare type of malignant disease with an incidence of 2 persons for one million population per year (Griswold et al., 1955; Ferber et al., 1962; Clemmesen, 1965). As evi- denced by data from the third National Cancer Sur- vey of the National Cancer Institute, only 37 cases of adrenal cortical carcinoma were recorded among the 163,000 cancer patients surveyed, accounting for 0.023% of all malignant tumours (King & Lack, 1978). Complete surgical extirpation remains the most effec- tive therapy with removal of the entire tumour and all extensions, if possible (Lewinsky et al., 1974; Hajjar et al., 1975; Greenberg & Marks, 1978; King & Lack, 1978). Clinical and/or biological signs of relapse may occur after the removal of the tumour and are most often referred to a local recurrence of the adrenal cor- tex carcinoma. However, it has to be pointed out that in most cases-70 to 95% according to the series-the patients decease with widespread metastatic disease (Lipsett et al., 1963; Hutter & Kayhoe, 1966; Schtein- gart et al., 1968; Harrison et al., 1973; Hajjar et al., 1975; King & Lack, 1978). The question thus arises as to whether metastases possess any hormonal activity. It is our purpose to present in vitro studies on steroid biosynthesis by a metastasis from a human primary adrenocortical carcinoma.
MATERIALS AND METHODS
Patient
A 26-year-old woman presented with characteristic symptoms and signs of hypercortisolism with weight gain, weakness, mild acne and hypertension. The patient had a round plethoric face, truncal obesity and hirsutism. Investi- gation led to a diagnosis of probable adrenocortical carci- noma which was confirmed at operation on the left side.
This was followed by complete remission of her symptoms and signs of Cushing’s syndrome. A treatment with o,p’-DDD (6 g a day), hydrocortisone (25 mg a day) and 9a-fluoro hydrocortisone (50 µg every day) was instituted after the removal of the carcinoma. However recurrence of the syndrome occured one year later. A second operation was performed and a large mass of adrenocortical carci- noma was removed again from the left side. The left kidney and the spleen were also removed. The same treatment as described above was pursued.
Six months later, a third operation was performed for removal of a large metastatic mass (1250 g) developed in the epiploon.
Histopathological findings
Microscopic examination of this epiploic mass revealed a tumoural proliferation with cellular pleomorphism, nuclear hyperchromasia, mitotic figures and showed necro- tic and hemorragic foci. These histologic features associ- ated with the tumour mass are known to be significant of malignant behaviour (Hough et al., 1979). It was concluded that this mass was a metastasis of the primitive adrenocor- tical carcinoma, but it was less differentiated than the in- itial tumour.
Experimental procedure
A part of this metastatic carcinoma was dissected free of adipose and connective tissues, trimmed and homogenized with a teflon-glass homogenizer in Earle’s medium buffer (Institut Pasteur, Paris), which was adjusted to 4 mM in calcium concentration to improve the steroid hydroxyl- ations. The incubations were carried out with one of these tritiated steroids (from New England Nuclear Corp.): 11-deoxycortisol (8.5 uCi; sp. act. = 44.1 Ci/mmol), pro- gesterone (8.4 µCi; sp. act. = 40-60 Ci/mmol) or 17-hyd- roxyprogesterone (7.8 uCi; sp. act. = 40.4 Ci/mmol). The radiochemical purity of these labelled steroids was checked by paper chromatography prior to use. The precursors were evaporated to dryness and taken up in 2.0 ml Earle’s solution, 30 min before the incubation started.
An NADPH generating system made up of NADP+ (1 mM; Merck Darmstadt, Germany) and malic acid (5 mM; Merck) neutralized with a 0.1 N potassium hydrox- yde solution was added to each incubation flask, to facili- tate the in vitro hydroxylations (Touitou et al., 1975). Each
* To whom requests for reprints should be addressed.
incubation was performed with 1.0 g, wet wt, of homogen- ized metastatic tissue in a final total volume of 10.0 ml. The incubations were carried out in triplicate under aerobic conditions in a Dübnoff metabolic shaking incubator at 37℃ for 2 hr. The enzymatic reactions were stopped with 15.0 ml acetone. To account for procedural losses during subsequent extraction and identification of synthetized steroids, trace amounts of [4-14C]cortisol (sp. act. = 55.4 mCi/mmol), [4-14C]-11-deoxycortisol (sp. act. = 50-60 mCi/mmol), [4-14C]cortisone (sp. act. = 59.8 mCi/mmol) or [4-14C]-11-deoxycorticosterone (sp. act. = 58.3 mCi/mmol) were added to the incubation flasks, according to the tritiated steroid precursor, to be used as internal standards. A more detailed description of incubation, filtration and extraction of steroids has been given in a previous paper (Touitou et al., 1978). Quantita- tive measurement of radioactivity was performed with a 3-channel liquid scintillation spectrometer (Tricarb, model 3255 Packard). Isotope contents were expressed as disinte- grations per minute (dpm). The results were calculated as percentage conversion (± SE) of radioactivity added as a substrate. The data were corrected for losses.
The following solvent systems were used for paper chromatography (PC) of labelled steroids and conversion products: PC 1 = dichloroethane-ethylene glycol; PC 2 = methylcyclohexane-toluene-formamide (1:1, v/v); PC 3 = benzene-formamide; PC 4 = hexane-benzene-propy- lene glycol (1:1, v/v); PC 5 = benzene-heptane-methanol- water (67:33:80:20, v/v); PC 6 = benzene-methanol-water (10:5:5, v/v). Individual isolated metabolites were further run in suitable chromatographic systems in which their isopolarity with carbon-14 internal standards was estab- lished. A conversion product was considered pure when a constancy of 3H:14C ratios was established in successive chromatographic systems. Each area corresponding to the studied steroids was characterized and evaluated by double isotope dilution using the 14C-labelled steroids as stan- dards. The methods of isolation, purification and charac- terization of cortisol and cortisone were previously de- scribed (Touitou et al., 1978, 1979). 11-Deoxycortisol was first separated from 17-hydroxyprogesterone added as a precursor in PC 2 system for 8 hr; finally it was character- ized by oxidation with chromic acid to androstenedione, the polarity of which was checked in PC 2 and PC 5 for 4 hr. 11-Deoxycorticosterone and 17-hydroxyprogesterone were separated from most of the steroids synthesized from progesterone as precursor in PC 2 system. As they were located in the same area in this system, PC 4 system for 7 hr was used to separate them. Deoxycorticosterone was then acetylated with unlabelled acetic anhydride into its 21-acetate which polarity was checked in PC 2 and PC 4 systems. 17-Hydroxyprogesterone was characterized by chromic acid oxidation into 44-androstenedione the polar- ity of which was checked as described above.
RESULTS
The biosynthesis of cortisol and cortisone (from 11-deoxycortisol as precursor), cortisol and 11-deoxy- cortisol (from 17-hydroxyprogesterone), 11-deoxycor- ticosterone, 17-hydroxyprogesterone and 11-deoxy- cortisol (from progesterone) was studied by in vitro incubations of a metastatic tissue of an adrenal corti- cal carcinoma.
Of all the studied steroids, three were mainly syn- thesized by the metastatis = cortisol from 11-deoxy- cortisol, deoxycorticosterone from progesterone and 11-deoxycortisol from 17-hydroxyprogesterone with conversion rates reaching respectively 24.9, 66.9 and 15.1% of the added precursor. This indicates the pres- ence of an important activity of the 118-hydroxylase
and 21-hydroxylase. By contrast, 17-hydroxylase ac- tivity was low, when compared, as only 0.42% of pro- gesterone was transformed by the metastasis into 17-hydroxyprogesterone. The synthesis of cortisone from 11-deoxycortisol was 1.5%.
Table 1 displays the data which are all expressed as the mean (± SE) of triplicate incubations.
DISCUSSION
Adrenocortical carcinomas have a bad prognosis. Even after surgical removal of the tumour, followed by treatment with o,p’-DDD an inhibitor of adrenal steroidogenesis, total recovery is seldom obtained. The relapse which usually occurs with both clinical and biological symptoms, has been most frequently related to an in situ recurrence of the tumour. How- ever, metastatic dissemination is frequent in adrenal carcinoma (Hutter & Kayhoe, 1966; Harrison et al., 1973; Greenberg & Marks, 1978; Hough et al., 1979) and the hypothesis that a secreting metastasis can be responsible for the relapse has been suggested in 1952 by Rapaport et al. (1952).
But as only surgery can completely eliminate the existence of a recurrence in situ of the carcinoma, the diagnosis of secreting metastasis is difficult to estab- lish. Histopathological study of the metastases does not help, in view of the absence of correlation between histological analysis and secretory function of the tumour (King & Lack, 1978).
In vitro incubation of metastases is therefore the only possible and absolute proof of their secreting capacities. Data presented in this paper clearly dem- onstrate the biosynthetic ability of this metastasis which metabolized all three precursors used : 11-deoxy- cortisol, progesterone and 17x-hydroxyprogesterone. The 11ß-hydroxylation, which is a characteristic of the adrenal cortex, is fairly high (25% conversion of 11-deoxycortisol to cortisol) and significantly higher than that obtained by incubation of “normal” human adrenals (Touitou et al., 1978), but still lower than that obtained with the primary tumour from the same patient as cortisol synthesis reached 64% (Touitou et al., 1979). This difference could be related to the treat- ment with o,p’-DDD (18 months, 6g a day, total dose: 3390 g) preceeding the removal of the metastasis as it is now established that o,p’-DDD inhibits 118-hydroxylase of human adrenal cortex (Touitou et al., 1978, 1979).
Other remarkable points are the high conversion rates of 17x-hydroxyprogesterone to 11-deoxycortisol and of progesterone to 11-deoxycorticosterone dem- onstrating the presence of a highly active 21-hydroxy- lase in the tissue. The results here obtained thus clearly show that the metastatic tissue possesses a steroid biosynthetic activity in vitro. The experimental data of Fraser et al. (1968) who showed a high syn- thesis of corticosterone by a metastatic tumour are in good agreement with this work.
However, this study led us to the following con- siderations: (a) despite histological differences between the primary tumour (well differentiated cells) and the metastasis (poorly differentiated cells), the lat- ter synthesized steroids in large amounts as did the primary tumour. This points out the lack of corre- lation between histological features and functional
| Precursor = S* | Cortisol | Cortisone | ||
|---|---|---|---|---|
| Percent | pmol | Percent | pmol | |
| Metastasis | 24.9 ± 2.9 | 17.4 ± 2.0 | 1.5 ±0.2 | 1.07 ± 0.14 |
| Primitive carcinoma | 64.4 ± 2.3 | 44.9 ± 1.6 | 7.3 ± 1.05 | 5.07 ± 0.70 |
Precursor = 17 OHPt
11-Deoxycortisol
Cortisol
| Metastasis | Percent | pmol | Percent | pmol |
|---|---|---|---|---|
| 15.1 ± 3.9 | 12.2 ± 3.1 | 3.9 ± 1.0 | 3.1 ± 0.8 |
Precursor = P#
Deoxycorticosterone
17a-Hydroxyprogesterone
11-Deoxycortisol
| Percent | pmol | Percent | pmol | Percent | pmol | |
|---|---|---|---|---|---|---|
| Metastasis | 66.9 ± 3.6 | 80.5 ± 4.4 | 0.42 ± 0.07 | 0.54 ± 0.14 | 0.44 ± 0.14 | 0.49 ± 0.14 |
* S = 11-deoxycortisol.
+ 17-OHP = 17-hydroxyprogesterone.
# P = progesterone.
The incubations were carried out with 1.0 g wet weight of homogenized metastatic tissue and [1,2-3H] 11-deoxycortisol, [1,2-3H] 17-hydroxyprogesterone or [1,2-3H] progesterone. Each flask contained: NADP+ (1 mM), malic acid (5 mM) in a total volume of 10.0 ml. Incubation lasted 2 hr at 37°C. Data are the mean ± SE of triplicate incubations.
status; (b) the metastasis studied in this report seems to present the same hormonal pattern as the primary carcinoma. However, we could recently observe a large difference in the in vitro synthesis of cortisol from 11-deoxycortisol as a precursor between an adrenocortical carcinoma resected from a 30-year-old woman, not preoperatively treated, and its liver metastasis. The respective conversion rates were 25% (primary carcinoma) and less than 0.5% (liver metas- tasis) (unpublished results). From a general point of view, the possible occurrence, as described for local recurrences of adrenocortical carcinomas (Rapaport et al., 1952), of metastases with a different hormonal secretion or no secretion at all cannot be ruled out.
Thus, in conclusion, systematic studies of changes in levels of circulating steroids in patients with adre- nal cortex carcinoma should take into account ca- pacity of metastases to alter production of steroids with time.
REFERENCES
CLEMMESEN J. (1965) Statistical studies in the aetiology of maglignant neoplasm. Danish Cancer Registry under the National Anticancer League. II. Munksgaard, Copenha- gen.
FERBER B., HARDY V. H., GERHARDT P. R. & SOLOMON M. (1962) Cancer in New-York State, Exclusive of New York City, 1941-1960. Bureau of Cancer Control, New York. State Dept of Health, Albany.
FRASER R., JAMES V. H. T., LANDON J., PEART W. S., DAW- SON A., GILES C. A. & MCKAY A. M. (1968) Clinical and biochemical studies of a patient with a corticosterone- secreting adrenocortical tumour. Lancet 2, 1116-1120.
GREENBERG P. H. & MARKS C. (1978) Adrenal cortical car- cinoma: a presentation of 22 cases and a review of the literature. Am. Surg. 44, 81-85.
GRISWOLD M. H., WILDER C. S., CUTLER S. J. & POLLACK E. S. (1955) Cancer in Connecticut, 1935 1951. Connecti- cut State Dept of Health, Hartford.
HAJJAR R. A., HICKEY R. C. & SAMAAN N. A. (1975) Adre-
nal cortical carcinoma. A study of 32 patients. Cancer 35, 549-554.
HARRISON J. H., MAHONEY E. M. & BENNETT A. H. (1973) Tumors of the adrenal cortex. Cancer 32, 1227-1235.
HOUGH A. J., HOLLIFIELD J. W., PAGE D. L. & HARTMANN W. H. (1979) Prognosis factor in adrenal cortical tumors. A mathematical analysis of clinical and morphological data. Am. J. clin. Path. 72, 390-399.
HUTTER A. M. & KAYHOE D. E. (1966) Adrenal Cortical Carcinoma. Clinical features of 138 patients. Am. J. Med. 41, 572-580.
KING D. R. & LACK E. E. (1978) Adrenal cortical carci- noma. A clinical and pathologic study of 49 cases. Cancer 44, 239-244.
LEWINSKY B. S., GRIGOR K. M., SYMINGTON T. & NEVILLE A. M. (1974) The clinical and pathologic features of non- hormonal adrenocortical tumors. Report of twenty new cases and review of the literature. Cancer 33, 778-790.
LIPSETT M. B., HERTZ R. & Ross G. T. (1963) Clinical and pathophysiologic aspects of adrenocortical carcinoma. Am. J. Med. 35, 374-383.
RAPAPORT E., GOLDBERG M. B., GORDAN G. S. & HINMAN F. (1952) Mortality in surgically treated adrenocortical tumors. Review of cases reported for the 20 year period 1930-1949, inclusive. Postgrad. Med. 11, 325-353.
SCHTEINGART D. E., OBERMAN H. A., FRIEDMAN B. A. & CONN J. W. (1968) Adrenal cortical neoplasms producing Cushing’s syndrome. A clinical pathologic study. Cancer 22, 1005-1013.
TOUITOU Y., BOGDAN A., LEGRAND J. C. & DESGREZ P. (1975) Aminoglutethimide and glutethimide effects on 18-hydroxycorticosterone biosynthesis by human and sheep adrenals in vitro. Acta Endocr. 80, 517-526.
TOUITOU Y., BOGDAN A. & LUTON J. P. (1978) Changes in corticosteroid synthesis of the human adrenal cortex in vitro, induced by treatment with o,p’-DDD for Cushing’s syndrome: evidence for the sites of action of the drug. J. steroid Biochem. 9, 1217-1224.
TOUITOU Y., BOGDAN A., AUZEBY A. & DOMMERGUES J. P. (1979) Glucocorticoid and mineralocorticoid pathways in two adrenocortical carcinomas: comparison of the effects of o,p’-dichlorodiphenyldichloroethane, aminoglu- tethimide and 2-p-aminophenyl-2-phenylethylamine in vitro. J. Endocr. 82, 87-94.