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Transforming growth factor-α and human cancer
Biomed. & Pharmacorher., 43 (1989) 651-660 0 Elsevier, Paris
g growth factor-a a
man cancer
J. YEH ‘* and Y.-C. YEH’
’ Department of Obstetrics, Gynecology and Reproductive Biology. Brigham and Women’s .Hos,oi’tai,and Department of Neurology. Children’s Hospital, Harvard M-d! 0 ‘ca!School 7.5Francis Street, Boston. MA 02115, USA: and ’ Department of Biochemistry and Molecular Biology. University of Arkansas for Medical Sciences. 4301 West Markham, Little Rock, AR 72205, USA (Received 21 August 1989; accepted 4 September 1989)
Summary Human transforming growth factor-alpha TGF-a, a polypeptide growth factor which causes reversible transformation of normal cells, is composed of 50 amino acid residues, has a 30 to 40 % amino acid homology to epidermal growth factor (EGF), and binds the EGF receptor. In human cancers, studies are beginning to show that TGF-a! could serve as a tumor marker and as a marker for the malignant potential of a tumor. Thus far, the types of carcinomas with which abnormal TGF-(r expression has been associated include liver, gastrointestinal, breast, skin, lung, brain and ovarian cancers. TGF-a may play a role in the processes involved with tumor initiation and tumor growth. In cell lines, TGF-a! has been found to be associated with autocrine and paracrine types of cellular growth initiation and with increased levels of oncogene expression. In summary, the evidence concerning human TGF-a are that TGF-a could serve as a marker for human cancers and that pn understanding of the basic actions of TGF-a couid help to explain the self-sustaining nature of tumors.
transforminggrowthfactor-a / cancer / tumor marker R&urn&
Fect!wa de croissance transformant alpha humain TGF-a et cancer.Le facteur de croissance transformant u humain TGFcr, facteur de croissance polypeptidique provoquant une transformation rdversible des cellules normales. se compose de SO acides amin& I1 montre une homologie de 30 ci 40 96 d’acides aminhs avec le facteur de croissance e’pidermique(EGF) et est capable de sefixer sur le rPcepteur de ce dernier. Dans les cancers humoins. des etudes sont actuellement entreprises pour montrer que le TGF-crpourrait servir de marqueur tumoral et de marqueur de malignith. Four I’instant, parmi les carcinomes associPs li une expression anormnle de TGFu on a trouvP les cancers du foie, ceux du tractus gastro-intestinal. du sein, de la peau. du poumon et de l’ovaire. Le TGFu joue. suns doute. un r6le dans ie processus mis en wvre lors de l’initiation de la tumeur et lors de sa croissance. Sur des l&&es cellulaires on a consrate’ que le TGF-ct s’accompagnait d’une initiation de la croissance des cellules du type autocrine et paracrine ainsi que d’une PIPvation du niveau d’expression des oncogbes. En r&urn&.ce qui ressort d propos du TGFa humain est qu’il pourrait e^t.reutilisP comme marqueur de cancers humains et quirne comprt!hension plus intime des effets de ce farteur de croissance permettrart de mieux expliquer le caractPre auto-entretenu des tumeurs.
faeteur de croissance rransfonnant-a /cancer ! marqueurtumoral
* Correspondence and reprints.
J. Yeh and Y.-C. Yeh
De Larco and Todaro [l I] were the first to isolate growth promoting factors which cause reversible morphologic transformation of normal cells. Subsequently, the peptide growth factors were divided into two major groups, transforming growth factor-alpha (TGF-cr) and transforming growth factor-beta (TGF-B). TGF-cr is a functional and structural analog of epidermal growth factor (EGF). TGF-b has been studied extensively and it has been found that TGF-b is a member of a large family of polypeptides with numerous physiological functions [29]. Qne important TGF-a, characteristic is its ability to transform cells. The most widely used test for this is reversible transformation of normal rat kidney (NRK) cells. The addition of TGF-a! to NRK cells in soft agar causes anchorage independent colony formation of the rat kidney cells, with the effects being reversible with the removal of the growth factor from the medium. Full transformation activity requires the presence of TGF-b [lo]. A second important characteristic of TGF-a is its ability to bind to EGF receptors. After TGF-a b!nds to the 170-kDa glycoprotein EGF receptor, it activates the intrinsic tyrosine kinase, then receptor autophosporylation, internalization and downregulation occurs [28]. Downward cf al. jl3] have demonstrated the homology between the EGF receptor and the oncogene protein v-erb-B. This similarity between the receptor which recognizes TGF-a! and an oncogene protein suggests that an abnormal level of the growth factor receptor may lead a signal to proliferate. Ozanne er al. [35] have substantiated this possibility by showing that human squamous carcinomas have 2- to IO-fold elevated levels of EGF receptors compared to normal skin and that the epidermoid carcinoma cell line A431 has elevated mRNA for EGF receptor and an amplification of the gene for the EGF receptor. cDNA of human TGF-a has been cloned and the nucleic acid and the peptide sequence of TGF-a have begun to be characterized. Demyck [12] reports that the gene for human TGF-cr is 70-100 kb and the TGF-a mRNA is ~4.5 kb. The human TGF-ar gene has been localized by in situ hybridization to human chromosome 2~13 [5, 451. Hayward et al. [ 181and Murray et al. [33] have found restriction fragment length polymorphism for the human TGF-a gene, with the restriction
endonucleases BamH I, Rsa I and Taq I recognizing the polymorphisms. The propeptide produced from translation of human TGF-a! mRNA is 160 amino acids in length. The TGF-cw propeptide is glycosylaled and secreted by the cells which produce them and is processed like other secreted polypeptides, with amino acid positions 38 and 88 in the propeptide acting as sites for further processing [24]. The mature peptide has a final length of 50 amino acids and 30-40 % amino acid homology to EGF. There are 6 cysteine residues, with the 3 resultant disulfide bond positions conserved between TGF-cr: and EGF. The secondary structure of TGF-a: in solution has been studied by Montelione et al. [31] using NMR spectroscopy. In the mature TGF-a peptide, the authors found 2 antiparallel beta-sheets. If one takes into account the structural constraints required by the 3 disultide bonds, along with the anti-parallel sheets, the secondary structure of human TGF-cu is similar to the secondary structure of human EGF. However, TGF-a has much faster amide proton/ deu;,ron exchange than EGF, suggesting that the structural dynamics of TGF-ar differs from that of EGF. Physiologically, TGF-ol is functionally analogous to EGF. Like EGF, subcutaneous TGF-a! administration induces a dose-response-related acceleration of eyelid opening and incisor eruption in newborn mice [43]. In addition, TGF-a! administration retards hair growth and body weight increase in the newborn mice. TGF-(w also has been found to be a potent in vivo angiogenic factor, more potent than EGF, with the hamster cheek pouch bioassay, which measures the ability of a substance for angiogenic activity [39]. Since one major characteristic of TGF-m is the ability of the peptide to transform cells, TGF-a! has been investigated for its role in human cancers. Sherwin et al. [40] analyzed the urine of 22 patients with disseminated cancer (5 with breast cancer, 5 with lung cancer, 5 with colon cancer, 4 with malignant melanomas, 2 with sarcomas and 1 with ovarian cancer). They found that 18 of 22 cancer patients had a high molecular weight peptide which had TGF-a-like activity while only 5 of 22 controls had the same activity in the urine. High performance liquid chromotography (HPLC) analysis of urines of cancer patients by Kimball ef al. [22] showed that the cancer patient urines contained 5 separate peaks of TGF-a-like activity, 2 of which were more elevated in the cancer patients than in the controls. More recently, Arteaga et al. [l, 161 mea-
Transforming growth factor* sured the pleural or ascitic effusions from 130 patients with different types of cancers. They found that 42 % of the effusions contained TGF-a (compared to 18 % of noncancer effusions), with statistical correlations between the presence of TGF-(Y and the number of metastic sites and the presence of TGF-cr and survival. Thus, in the preliminary studies, it appears that the presence of TGF-cu is associated with human cancers. Below, we review the findings regarding TGF-a! in individual human cancers. TGFcl in liver cancer In human hepatic cancer, TGF-a! in the urine is associated with the presence of tumor. Rat hepatic carcinoma cells appear to produce large quantities of TGF-a. Additionally, TGF-ar levels are increased with liver regeneration, suggesting that this growth factor is associated with active cellular division of hepatic cells. Human hepatoma We studied 31 patients with hepatocellular carcinoma, 15 with probable hepatic carcinoma, 4 with metastatic liver cancer and 33 age- and sexmatched controls [47] and found that TGF-(r in the urine is significantly higher in the patients with cancer. In the hepatocellular cancer patients, was concentration urinary TGF-(w 2 1.89 + 21 .OOpg/g creatinine. In the non-cancerous controls, the mean TGF-cr concentration was 4.94 f 2.77 pg/g creatinine (P< 0.05). There was no difference in the urinary EGF concentrations between the two groups. In iiiis study, we also compared the sensitivity of urinary TGF-a to serum a-fetoprotein to detfect hepatocellular carcinoma. Alpha-fetoprotein alone detected 58.7 % of the carcinomas. TGF-a was more sensitive as a marker and detected 7 1.7 % of the cancers. The combined sensitivity of using both tests for detecting liver cancer was 93.5 %. Thus, TGF-ar could effectively serve as another marker to detect and monitor progress of patients with hepatocellular carcinomas. Rat hepatocellular carcinoma cells Rat hepatoma cells produce and secrete the mature TGF-a! peptide. The rat hepatocellular cell line JMl was originally established from a liver tumor in a Fisher 344 rat given the carcinogen diethylnitrosamine. Northern blotting of RNA from JMl cells show that the cells produce a 4.5 kb TGF-a! mRNA [24]. Studies using HPLC, radioimmunoassay and Western blotting of the
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JMl medium reveal that TGF-a! and its precursor peptide are produced in significant quantities. This study confirms in vitro that hepatocellular cancer cells can produce large amounts of TGF-a and underlines the importance of our studies of urinary TGF-ar excretion in humans with hepatocellular carcinoma [47]. Liver regeneration Liver regeneration represents another model system for studying factors regulating rapid cellular proliferation. Mead and Faust0 [30] performed partial hepatectomies in rats to examine the factors involved in liver regeneration. They found that TGF-(r expression increased after partial hepatectomy. The mRNA for TGF-a! was undetectable in the liver until 12 h after surgery, and then the TGF-c~ mRNA concentrations peaked at 24 h after surgery, coincident with the peak of cellular division. The TGF-a mRNA levels in the hepatocytes increased 9-fold between the beginning of detectable TGF-a levels and peak levels. In primary liver cell cultures, the addition of TGF-a! was associated with a 13-fold elevation of DNA synthesis. Moreover, in the cells undergoing rapid DNA synthesis, the level of TGF-a! mRNA increased. In this model system of rapid cellular proliferation, TGF-(r acts as an autocrine or paracrine factor. This study may help to explain the self-activating nature of the elevations of TGF-a! production in human hepatomas, once the initiation events for cancer growth occur. TGFcl and gastrointestinal cancer TGF-cr is produced by both normal and cancerous gastrointestinal (GI) tissues. The level of TGF-a! production in cancers and the role of TGF-a! in GI cancers are currently under active investigation. Gastrointestinal tissues and tumors In gastric tumors, TGF-(U production may be elevated. In a series of 38 gastrectomies 121,26 of which were for gastric cancers, 63 separate gastric biopsy specimens were obtained. TGF-(U mRNA was present in both cancerous and noncancerous specimens. In 16 paired specimens, where both cancerous and noncancerous tissues were obtained from the same patient, TGF-a mRNA was found to be present in higher quantities in the cancerous regions. In a very small study 1261, Northern blot analysis was used to investigate biopsies of human gastrointestinal tract from the esophagus to the colon. The investigators de-
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tected the presence of TGF-a! mRNA, after p01y A+ purification, from all parts of the normal intestinal tract, with the highest abundance in the duodenum. In 4 patients with primary colon cancers, TGF-a mRNA was also detected, albeit in quantities which were similar to that observed in normal tissues. In two patients with gastric carcinoma, they also did not detect a difference in the mRNA levels between the normal and cancerous tissue. The conflicting data concerning TGF-a in human gastrointestinal cancers need to be resolved by studying larger series of patients before definitive statements can be made concerning the role of the growth factor as a marker for diagnosis or prognosis in gastrointestinal cancer. Cell lines Human colon cancer cell lines produce TGF-ol. Coffey er al. [S] and Hanauske et al. [ 171measured TGF-ar production by human colon cancer cell lines and found that a majority of cell lines were able to produce TGF-a. In addition, the colon cell lines produce the receptor for TGF-a [8]. Buick et ai. 161studied the transformation of rat intestinal epithelium with the oncogene H-r-as. The oncogene transformation of the ceils was associated with an overexpression of TGF-rw mRNA. This overexpression of mRNA by transformed cells is consistent with the in virro Endings of elevated TGF-cr production by the colon cancer cell lines. TGFa in breast cancer Breast cancer is one of the commonest forms of cancer in American women. The mediators of breast cancer growth other than estrogen are still not well understood. TGF-a-like activity has been detected in normal breast tissue [36] and in human breast milk [49]. An abnormal production of TGF-a may, therefore, be associated with breast cancer growth. Human breast cancer : TGFc( gene expression In understanding the role of TGF-a in human breast cancer tissue growth, two important questions are : (1) is there an association between TGF-a expression and breast cancer ? and (2) is there a relationship between TGF-a expression and the presence of estrogen receptors ? Three studies have been published recently which attempt to address these questions. Travers ef al. 1441 studied TGF-a mRNA expression of 52 malignant breast biopsies and 15 nonmalignant tumors of the breast and 4 normal breast specimens. They detected TGF-a mRNA in 38 % of the
breast cancer tissues and in 22 % of normal breast biopsies. They also studied the expression of the TGF-a! receptor, the EGF receptor, in the breast specimens and found EGF receptor mRNA expression in 42 % of 45 cancer specimens while it was detectable in 100 % of 15 benign specimens. Macias et al. [25] used EGF-binding competition as their assay for TGF-a! activity and found that 31% of 45 primary mammary carcinomas had measurable TGF-a! activity. They did not use a normal group for comparison. In a small study, Perroteau et al. [36] measured the immunoreactive TGF-a activity in breast biopsies. In their series of 22 primary breast cancer biopsies and 4 noncancerous breast biopsies, all the specimens contained TGF-rr activity by radioimmunoassay, but there was no difference in the levels of TGF-(r in the cancerous specimens compared to the 4 normals. Increased TGF-cr expression may occur in breast cancers, but the degree and the type of overexpression still needs to be determined. The relationship between the presence of estrogen receptor and TGF-(w in breast cancer is yet unresolved. Travers et al. [44] found that TGF-a! mRNA and the mRNA for the TGF-cw receptor, the EGF receptor, were more than twice as common in the breast cancers which were negative for estrogen receptors. In contrast, Macias et al. [25] and Perroteau et af. [36] found no association between the estrogen receptor content of breast tumors and TGF-a levels. Urinary excretion of TGFcr The presence of TGF-ar in the urine of patients with disseminated breast cancers has been documented by Stromberg el al. 1421. They collected 22 I of urine from 3 patients with disseminated breast cancer and by chromatography, EGF-receptor competition and TGF-ar radioimmunoassay, they demonstrated that TGF-a was present in the urine of these patients. It would be useful to extend their studies to see if TGF-cr could serve as a tumor marker for diagnostic or prognostic purposes. Breast cancer cell lines In breast cancer cell lines, TGF-ar appears to stimulate cellular growth. Using the human mammary tumor cell line ZR-75-1, Novak-Hofer et at. [34] measured the relationship between TGF-a! and the phosphorylation of S6, a ribosomal protein. They showed that TGF-cw increases cell growth in this mammal cell line and phosphorylates the S6 kinase. In addition, they
Transforming growth factor* showed that the mechanism for the TGF-cu mediated increase in cell number is probably different from the mechanism for the increase in cell number by estradiol-there was no change in the level of S6 phosphorylation with estrogen stimulated cell growth. Bjorge et al. [4] have studied the human breast cancer cell line MDA468. They found that phorbol ester and EGF stimulate the production of mRNA for TGF-a and its receptor, the EGF receptor. They concluded that this growth factor could serve as an autocrine type of a signal to the cell line. Valverius ef al. [46] studied normal tissue from breast reduction mammoplasties and transformed the normal tissues with several combinations of carcinogenic drugs. In contrast to the other studies, they found that mRNA for TGF-a was no different between the normal cells and the immortalized cells derived from the normal cells. However, their findings do not necessarily reflect the situation in vivo, where the transformation process to cancer is probably different and where breast cancer cells could behave differently from in vitro. TGF-cl in melanoma and skin diseases With the similarities between TGF-a and EGF, it would be consistent for TGF-a to have an effect on the human epidermis. TGF-a is present in normal human skin. Coffey et al. [7] found that primary cultures of human keratinocytes produced TGF-a mRNA. In addition, incubations of these cells with TGF-a increased TGF-a mRNA production, suggesting the potential for auto-induction for these cells. In normal skin biopsies, in situ hybridization and immunohistochemistry showed the presence of TGF-a mRNA and protein. Melanoma TGF-a has been isolated from the urine of patients with melanoma. Sherwin er al. [40] found high molecular weight TGF-a in the urine of patients with disseminated melanoma. Using 3 I of urine from patients with melanoma, Kim et al. [21] isolated and purified by 200 OOO-fold a low molecular weight (4545 Da) form of TGF-a from the urine of these patients. Melanoma cell lines produce TGF-a. Marquardt and Todaro [27] found that the human metastatic melanoma cell line A2058 produced TGF-a. Richmond et al. [37] found that the human malignant melanoma cell line Hs0294
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produced TGF-cu. Ellem et al. [I51 tested 5 melanoma cell lines and found that all responded to ultraviolet radiation (UVR) with production of TGF-a. They found that a 12-h exposure of normal foreskin melanocytes to UVR resulted in the cellular production of TGF-a. Thus, they hypothesized that UVR resulted in the production of TGF-a, which subsequently is involved in the autocrine induction of abnormal melanocyte response. This finding may explain the origin of abnormal melanocyte cell growth, leading to the development of melanoma. Restriction fragment length polymorphism of the human TGF-a gene found in melanoma cell lines and melanoma biopsy specimens may eventually be used to identify susceptible tissues for melanoma. Using the restriction endonuclease Taq I to analyze the DNA, Hayward et al. [19] found that a 2.7 kb fragment was associated with melanoma cell lines. A large series showing a statistical correlation for the association in human melanoma would be beneficial in this regard. Psoriasis Epidermal hyperproliferation occurs in psoriasis, a common benign disease of the epidermis. Elder et al. [14] studied TGF-a expression in biopsies of psoriatic epidermis, normal-appearing skin of patients with psoriasis and normal skin. They used scanning densitometry of Northern blots to measure the TGF-a mRNA in these skin specimens. Psoriatic TGF-a mRNA was increased 4.4-fold in total mRNA preparations and 5.7-fold in poly A+ RNA preparations when compared to normal controls. In their measurements of the TGF-a peptide obtained by acid-ethanol extraction of the cells, they found a 6-fold increase in the TGF-a protein content of the psoriatic cells. Thus, it appears that TGF-a expression is a component that is actively increased in the hyperproliferative state of psoriasis. Cell cultures TGF-a was able to stimulate oncogene protein production in fibroblasts. Cutry ef al. [9] studied the fibroblast cell line C3H lOTI/ and found that TGF-a stimulated DNA synthesis. In addition, TGF-a causes a large increase in mRNA for two oncogenes, c-fos and c-myc, suggesting that one possible mechanism for the effects of TGF-a on skin cells, in addition to auto-induction, could be the stimulation of oncogene protein production.
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lGFu in lung cancer TGF-a has been detected in the urine and plural effusions of patients with lung cancer [I, 401. BY using cell lines, several additional observations have been made concerning lung cancer. Soderdahl et al. [41] found that TGF-a is expressed in 4 of 6 nonsmall cell human lung cancer cell lines. They found that TGF-a was expressed in a squamous-cell carcinoma, 2 adenocarcinomas and one large cell carcinoma cell line. Bergh PI extended this study and found that there was an association with TGF-a! production and the ability of the lung cancer cells to produce stroma fibrosis when the cancer cells were transplanted into nude mice. It then appears that TGF-a! expression may be correlated with some of the phenotypical characteristics of some lung cancer cell lines. By the use of monoclonal antibodies to human TGF-a., Imanishi et al. [20] demonstrated that the antibodies could inhibit lung cancer cell growth. They incubated an anti-human TGF-a monoclonal antibody with two different human lung adenocarcinoma cell lines (A-549 and PC-g). The growth of the cancer cells significantly decreased with the addition of the monoclonal antibody to the culture medium. This inhibitory effect was reversible with the addition of high levels of TGF-CL The significant implication from this study is that monoclonal antibodies could serve as a potential method for the control of lung cancer proliferation. TGFu in brain cancers Ex first brain tumor to be studied for TGF-a! was human glioma. Samuels et al. [38] used polyclonal antibodies to TGF-a to study benign and malignant human gliomas. They chose this particular tumor to study because of the direct relationship between the morphology of the tumor and malignancy. They found a strong relationship between the malignant nature of the tumors and the presence of TGF-a. In gliomas, therefore, TGF-a could serve as a protein marker for the degree of malignancy. TGFu in the genital and urinary tracts For ovarian cancer, Sherwin et al. [40] found elevated levels of TGF-LYin the urine of a patient with disseminated disease. Arteaga et al. [l] found e!evated levels of TGF-a in the ascitic effusions of 13 of 31 patients with ovarian cancer. Detailed reports concerning TGF-a in bladder cancer have yet to he published. Yura et al. [48], however,
studied the potential tumor enhancing effects of rat urine on rat urinary bladder cell growth. They measured omithine decarboxylase inducing activity, which is a characteristic of rodent skin tumor promoters, in the target rat bladder cancer cell line and found that TGF-a: and EGF have dose-response-related ability to induce this enzyme activity. Thus, TGF-a! as a component of the excretion products of urine could cause events leading to cancer formation in rats. Potential diagnostic and therapeutic use of TGF+x in cancer A summary of the relationship of TGF-(U to human cancer presented in this review is shown in Table I. Given these findings, one can speculate on the potential use of TGF-a! in cancer diagnosis and therapeutics. The situations in which abnormalities of TGF-a production have been noted could be used advantageously to serve as cancer markers. For example, TGF-a present in the urine of patients with hepatoma could be used as a measure of tumor load. Another example is the use of TGF-a in pleural or ascitic effusions to measure the extent of the disease. In addition, measurements of TGF-ar could be used in conjunction with other known markers of tumors, like a-fetoprotein and estrogen receptors, to increase the diagnostic techniques available to patients. In our laboratory (Yeh et al., submitted), TGF-a could be readily measured in small quantities of saliva. Therefore, in addition to using urine and bodily effusions to measure TGF-a! levels, saliva could be used to quantitate TGF-a! levels in cancer patients. Therapeutically, an understanding of the role of TGF-a could lead to treatment of human cancers. As presented in Table II, there are a number of mechanisms in which TGF-a could influence the growth of cancerous cells. Each of the potential sites of TGF-(r action could serve as a site to inhibit cancer growth, if abnormal TGF-(u activity were the cause of the cancer. One example of the use of this type of intervention is the work performed by Imanishi et al. [20] where monoclonal antibodies to TGF-cu could be administered to block carcinoma growth. Another possible method of treating cancer patients, given Samuel et al.3 [38] finding of the relationship of elevated TGF-a! levels in malignant human brain cancers, is that TGF-a could be used as a marker for targeted chemotherapy, with TGF-a serving as a marker for high concentrations of malignant cells.
Transforming
growth factora
and human cancer
657
Table I. TGF-a in human cancers. Human cancer Liver Intestinal
Breast
Findings Elevated TGF-a in urine of hepatoma
tract
Elevated Elevated pancreas Elevated
Ref:
patients
TGF-a mRNA in gastric cancers TGF-a in effusions of patients with colon, stomach and cancers TGF-a in the urine of colon cancer patients
Elevated TGF-a mRNA
in primary breast cancers
TGF-a in urine of breast cancer patients Elevated TGF-a in effusions of patients with breast cancer Skin
TGF-a
Lung
Elevated TGF-a
in effusions of patients with lung cancer
Brain Ovary
Elevated Elevated Elevated Elevated
in urine of lung cancer patients associated with malignant forms of gliomas in urine of patient with ovarian cancer in effusions of patients with ovarian cancer
in urine of melanoma
TGF-a TGF-a TGF-a TGF-a
Table II. Potential mechanisms in human cancers.
patients
for the role of TGF-a
Mechanism Abnormal elevation of TGF-a protein levels Loss of control of TGF-a gene regulation Abnormal amplification of TGF-a gene number Change in paracrine or autocrine control of TGF-a production Change in receptor number of receptor gene number for TGF-a Change in post-receotor effects of TGF-a Change in synergistic effects with other hormones, growth factors or oncogenes
Table III. Inducers of TGF-a Inducer Addition of TGF-a Gonadotropins Estrogens Progestins EGF Phorbol ester Ultraviolet radiation Regenerative state Oncogene transformation
Yeh et al.
[47l
Bennett et al.
[21
Arteaga et al. Sherwin et al. Travers et a!. Stromberg et al. Arteaga et al. Kim et al.
111
WI WI [42]
PI [211
Arteaga et al. Sherwin et al.
WI
Samuels et al.
[381
Sherwin et al. Arteaga et al.
WI
PI
111
!f e!evated TGF-a is a major factor in cancer cell growth, the knowledge concerning inducers of TGF-a! production presented in Table III couiJ be used to help prevent malignant growth. If the factors which lead to induction of abnormal TGF-a production in organs susceptible to cancer could be blocked, one may be able to hinder the cellular proliferative steps which could lead to malignancy. Blocking TGF-a! production, one after ultraviolet radiation could hypothesize, exposure in patients who are susceptibie to melanoma or blocking TGF-a! expression after estrogen and/or progesterone exposure in patients who are susceptible to breast cancer might reduce the susceptible patient’s risk of cancer formation or growth.
production.
Example Primary skin cultures Rat ovary Breast cancer cell line Breast cancer cell line Breast cancer cell line Breast cancer cell line Melanoma cell ‘line Liver regeneration Rat intestinal cells
ReJ Coffey ef a/. Kudlow et al. Perroteau et al. Murphy 8s Dotzlaw Bjorge et al. Bjorge et al. Ellem et al. Mead & Faust0 Buick et al.
171 1231 I361 ;:;I t41
WI
[301 PI
J. Yeh and Y.-C. Yeh
Conclusion In the decade since the first isolation and characterization of TGF-cu, significant steps have been made in elucidating the presence and potential roles of this growth factor in human cancers. Though detailed studies need to be performed, TGF-(w is already promising as a marker for several human cancers.
eferences Arteaga C.L., Hanauske A.R., Clark GM., Osborne C.K., Hazarika P., Pardue R.L., Tio F. & Von Hoff D.D. (1988) Immunoreactive a transforming growth factor activity in effusions from cancer patients as a marker of tumor burden and patient prognosis. Cancer Res. 48, 5023 Bennett C., Paterson I.M., Corbinshley C.M. % Luqmani Y.A. (1989) Expression of growth factor and epidermal growth factor receptor encoded transcripts in human gastric tissues. Cancer Rex 49, 2104 Bergh J. (1988) The expression of the platelet derived and transforming growth factor genes in human nonsmall lung cancer cell lines is re!ated to tumor stroma formation in nude mice tumors. Am. J. Pathol. 133, 434 Bjorge J.D., Payerson A.J. & Kudlow J.E. (1989) Phorbol ester or epidermal growth factor (EGF) stimulates the concurrent accumulation of mRNA for the EGF receptor and its ligand transforming growth factor-a in a breast cancer cell line. J. Biol. Chem. 264, 4021 Brissenden J.E., Derynck R. & Francke U. (1985) Mapping to transforming growth factor a gene on human chromosome 2 close to the breakpoint of Burkitt’s lymphoma t (2;8) variant translocation. Cancer Rex 45, 5593 Buick R.N., Filmus J. & Quaroni A. (1987) Activated H-ras transforms rat intestinal epithelial cells with expression of a-TGF. Exp. Cell Res. 170, 300 Coffey RJ., Derynck R., Wilcox J.N., Bringman T.S., Goustin A.S., Moses H.L. & Pittelkow M.R. (1987) Production and auto-induction of transforming growth factor-a in human keratinocytes. Nature 328, 817 Coffey R.J., Shipley G.D. & Moses H.L. (1986) Production of transforming growth factors by human colon cancer lines. Cancer Res. 46, I 164 Cutry A.F., Kinniburgh A.J., Twardzik & Wenner C.E. (1988) Transforming growth factor alpha (TGF-a) induction of c-fos and c-myc expression in C3H lOT1/2 cells. Biochem. Biophy. Res. Commun. 152, 216
IO Dart L.L., Smith D.M., Mevers C.A.. Snom M.B. & Frolik C.A. (1985) Transforming growth factors from a human tumor cell : characterization of transforming growth factor /I and identification of high molecular weight transforming growth factor-a. Biochemistry 24, 5925 I1 De Larco J.E. & Todaro G.J. (1978) Growth factors from murine sarcoma virus-transformed cells. Proc. Natl. Acad. Sci. USA 75, 4001 12 Derynck R. (1988) Transforming growth factor Q. Ce!! 54. 593 13 Downward J., Yarden Y., Mayes E., Scrace G., Totly N., Stockwell P., Ullrich A., Schlessinger J. & Waterfield M.D. (1984) Close similarity of epiderma1 growth factor receptor and v-erb-B oncogene protein sequences. Nature 307, 521 14 Elder J.T., Fisher G.J., Lindquist P.B., Bennett G.L., Pittelkow M.R., Coffey R.J., Ellingsworth L., Derynck R. & Voorhees J.J. (1989) Overexpression of transforming growth factor-u in psoriatic epidermis. Science 243, 811 15 Ellem K.A.O., Cullinan M., Baumann K.C. & Dunstan A. (1988) UVR induction of TGF-a : a possible autocrine mechanism for the epidermal melanocytic response and for promotion of epiderma1 carcinogenesis. Carcinogenesis 9, 797 16 Hanauske A.R., Arteaga C.L., Clark G.M., Buchok J., Marshall M., Hazarika P., Pardue R.L. & Von Hoff D.D. (1988) Determination of transforming growth factor activity in effusions from cancer patients. Cancer 61, 1832 17 Hanauske A.R., Buchok J., Scheithauer W. & Von Hoff D.D. (1987) Human colon cancer cell lines secrete a TGF-like activity. Br. J. Cancer 55, 57 18 Hayward N.K., Nancarrow D.J. & Bell G. (1987) A Taq I polymorphism for the human transforming growth factor-a gene (TGFA). Nucdeic Acid Res. 15, 5503 19 Hayward N., Nancarrow D., Ellem K., Parsons P. & Kidson C. (1988) A TAQI RFLP of the human TGF-a! gene is significantly associated with cutaneous malignant melanoma. Ink J. Cancer 42, 558 20 lmanishi K.I., Yamaguchi K., Kuranami M.: Kyo E., Hozumi T. & Abe K. (1989) Inhibition of growth of human lung adenocarcinoma cell lines by antitransforming growth factor-a monoclonal antibody. J. Natl. Cancer Inst. 81, 220 21 Kim M.K., Warren T.C. & Kimball E.S. (1985) Purification and characterization of a low molecular weight transforming growth factor from the urine of melanoma patients. J. Biol. Chem. 260, 9237 22 Kimball E.S., Bohn W.H., Cockley K.D., Warren T.C. 8r Sherwin S.A. (1984) Distinct high-performance liquid chromatography pattern of transforming growth factor activity in urine of cancer patients as compared with that of normal individuals. Cancer Res. 44, 3613 23 Kudlow J.E., Kobrin M.S., Purchio A.F., Twardzik D.R., Hemandez E.R., Asa S.L. & Adashi E.Y.
Transforming growth factoru (1987) Ovarian transforming growth factor-a gene expression : immunohistochemical localization to the theta-interstitial cells. Endocrinology 121, 1577 24 Luetteke N.C., Michalopoulos G.K., Teixido J., Gilmore R., Massague J. & Lee D.C. (1988) Characterization of high molecular weight transforming growth factor a produced by rat hepatocellular carcinoma cells. Biochemistry27, 6487 25 Macias A., Perez R., Hagerstrom T. & Skoog L. (1989) Transforming growth factor-a in human mammary carcinomas and their metastases. Anticancer Res. 9, 177 26 Malden L.T., Novak U. dc Burgess A.W. (1989) Expression of transforming growth factor-a messenger RNA in the normal and neoplastic gastrointestinal tract. Znt.J. Cancer 43, 380 21 Marquardt H. & Todaro G.J. (1982) Human transforming growth factor. J. Biol. Chem. 257, 5220 28 Massague J. (1983) Epidermal growth factor-like transforming growth factor : II. interaction with epidermal growth factor receptors in human placenta membranes and A431 cells. J. Biol. Chem. 258, 13614 29 Massague J. (1987) The TGF-#I family of growth and differentiation factors. Ce!! 49, 437 30 Mead J.E. &cFaust0 N. (1989) Transforming growth factor a may be a physiological regulator of liver regeneration by means of an autocrine mechanism. Proc. Natl. Acad. Sci. USA 86, 1558 G.T., Winkler M.E., Burton L.E., 31 Montelinone Rinderknecht E., Spom M.B. & Wagner G. (1989) Sequence-specific ‘H-NMR assignments and identification of two small antiparallel fisheets in the solution structure of recombinant human transforming growth factor-a. Proc. Natl. Acad. Sci. USA 86, 1519 32 Murphy L.C. & Dotzlaw H. (1989) Endogenous growth factor expression in T-47D, human breast cancer cells, associated with reduced sensitivity to antiproliferative effects of progestins and antiestrogens. Cancer Res. 49, 599 33 Murray J.C., Buetow K.H. & Bell G.I. (1986) RFLPs for transforming growth factor-a (TGFA) gene at 2~13. Nucleic Acids Res. 14, 7136 34 Novak-Hofer I., Kung W., Fabbro D. & Eppenberger U. (1987) Estrogen stimulates growth of mammary tumor cells ZR-75 without activation of S6 kinase and S6 phosphorylation. Eur. J. Biochem. 164, 445 35 Ozanne B., Shum A., Richards C.S., Cassells D., Grossman D., Trent J., Gusterson B. & Hendler F. (1985) Evidence for an increase of EGF receptors in epidermoid malignancies. Cancer Cells 3, 41 34 Perroteau I., Salomon D., DeBortoli M., Kidwell W., Hazarika P., Pardue R., Dedman J. & Tam J. (1986) Immunological detection and quantitation of a-transforming growth factors in human breast carcinoma cells. Breast Cancer Res. Treat. 7, 201
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37 Richmond A., Lawson D.H., Nixon D-W. & Chawla R.K. (1985) Characterization of autosimulatory and transforming growth factors from human melanoma cells. Cancer Res. 45, 6390 38 Samuels V., Barrett J.M., Bockman S., Pan&is C.G. & Allen M.B. (1989) Immunocytochemical study of transforming growth factor expression in benign and malignant gliomas. Am. J. Pathol. 134, 895 _ 39 Schreiber A.B., Winkler M.E. $ Derynck R (1986) Transforming growth factor-a : a more potent angiogenic mediator than epidermal growth factor. Science 232, 1250 40 Sherwin S.A., Twardzik D.R., Bohn W.H., Cockley K.D. & Todaro G.J. (1983) High-molecular weight transforming growth factor activity in the urine of patients with disseminated cancer. Cancer Res. 43, 403 41 Soderdabl G., Betsholtz C., Johansson A., Nilsson K. & Bergh J. (1988) Differential expression of platelet-derived growth factor and transforming growth factor genes in small and non-small-cell human lung carcinoma lines. Znt.J. Cancer 41, 636 42 Stromberg K., Hudgins W.R. & Orth D.N. (1987) Urinary TGFs in neoplasia : immunoreactive TGF-a in the urine of patients with disseminated breasr carcinoma. B&hem. Biophys. Res. Commun. 144, 1059 43 Tam J.P. (1985) Physiological effects of transforming growth factor in the newborn mouse. Science 229, 673 44 Travers M.T., Barrett-Lee P.J., Berger U., Luqmani Y-A., Gazet J., Powles T.J. & Coombes R.C. (1988) Growth factor expression in normal, benign, and malignant breast tissue. Br. Med. J. 296, 1621 45 Tricoli J.V., Nakai H., Byers M.C., Rall L.B., Bell G.I. & Shows T.B. (1985) Assignment of the gene coding for human TGF-a to chromosome 2~13. Cyfol. Cell. Genet. 40, 762 46 Valverius E.M., Bates S.E., Stampfer M.R., Clark R., McCormick F., Salomon D.S., Lippman M.E. 8r Dickson R.B. (1989) Transforming growth factor a production and epidermal growth factor receptor expression in normal and oncogene transformed human mammary epithelial cells. Mol. Endocrinol. 3, 203 47 Yeh Y.C., Tsai J.F., Chuang L.Y., Yeh H.W., Tsai H.J., Florine D.L. & Tam J.P. (1987) Elevation of transforming growth factor a and its relationship to the epidermal growth factor and a-fetoprotein levels in patients with hepatocellular carcinoma. Cancer Res. 47, 896 48 Yura Y., Hayashi O., Kelly M. & Oyasu R. (1989) Identification of epidermal growth factor as a component of the rat urinary bladder tumor-enhancing urinary fractions. Cancer Res. 49, 1548 49 Zwiebel J.A., Bano M., Nexo E., Salomon D.S. & Kidwell W.R. (1986) Partial purification of transforming growth factors from human milk. Cancer Res. 46, 933
g growth factor-a a
man cancer
J. YEH ‘* and Y.-C. YEH’
’ Department of Obstetrics, Gynecology and Reproductive Biology. Brigham and Women’s .Hos,oi’tai,and Department of Neurology. Children’s Hospital, Harvard M-d! 0 ‘ca!School 7.5Francis Street, Boston. MA 02115, USA: and ’ Department of Biochemistry and Molecular Biology. University of Arkansas for Medical Sciences. 4301 West Markham, Little Rock, AR 72205, USA (Received 21 August 1989; accepted 4 September 1989)
Summary Human transforming growth factor-alpha TGF-a, a polypeptide growth factor which causes reversible transformation of normal cells, is composed of 50 amino acid residues, has a 30 to 40 % amino acid homology to epidermal growth factor (EGF), and binds the EGF receptor. In human cancers, studies are beginning to show that TGF-a! could serve as a tumor marker and as a marker for the malignant potential of a tumor. Thus far, the types of carcinomas with which abnormal TGF-(r expression has been associated include liver, gastrointestinal, breast, skin, lung, brain and ovarian cancers. TGF-a may play a role in the processes involved with tumor initiation and tumor growth. In cell lines, TGF-a! has been found to be associated with autocrine and paracrine types of cellular growth initiation and with increased levels of oncogene expression. In summary, the evidence concerning human TGF-a are that TGF-a could serve as a marker for human cancers and that pn understanding of the basic actions of TGF-a couid help to explain the self-sustaining nature of tumors.
transforminggrowthfactor-a / cancer / tumor marker R&urn&
Fect!wa de croissance transformant alpha humain TGF-a et cancer.Le facteur de croissance transformant u humain TGFcr, facteur de croissance polypeptidique provoquant une transformation rdversible des cellules normales. se compose de SO acides amin& I1 montre une homologie de 30 ci 40 96 d’acides aminhs avec le facteur de croissance e’pidermique(EGF) et est capable de sefixer sur le rPcepteur de ce dernier. Dans les cancers humoins. des etudes sont actuellement entreprises pour montrer que le TGF-crpourrait servir de marqueur tumoral et de marqueur de malignith. Four I’instant, parmi les carcinomes associPs li une expression anormnle de TGFu on a trouvP les cancers du foie, ceux du tractus gastro-intestinal. du sein, de la peau. du poumon et de l’ovaire. Le TGFu joue. suns doute. un r6le dans ie processus mis en wvre lors de l’initiation de la tumeur et lors de sa croissance. Sur des l&&es cellulaires on a consrate’ que le TGF-ct s’accompagnait d’une initiation de la croissance des cellules du type autocrine et paracrine ainsi que d’une PIPvation du niveau d’expression des oncogbes. En r&urn&.ce qui ressort d propos du TGFa humain est qu’il pourrait e^t.reutilisP comme marqueur de cancers humains et quirne comprt!hension plus intime des effets de ce farteur de croissance permettrart de mieux expliquer le caractPre auto-entretenu des tumeurs.
faeteur de croissance rransfonnant-a /cancer ! marqueurtumoral
* Correspondence and reprints.
J. Yeh and Y.-C. Yeh
De Larco and Todaro [l I] were the first to isolate growth promoting factors which cause reversible morphologic transformation of normal cells. Subsequently, the peptide growth factors were divided into two major groups, transforming growth factor-alpha (TGF-cr) and transforming growth factor-beta (TGF-B). TGF-cr is a functional and structural analog of epidermal growth factor (EGF). TGF-b has been studied extensively and it has been found that TGF-b is a member of a large family of polypeptides with numerous physiological functions [29]. Qne important TGF-a, characteristic is its ability to transform cells. The most widely used test for this is reversible transformation of normal rat kidney (NRK) cells. The addition of TGF-a! to NRK cells in soft agar causes anchorage independent colony formation of the rat kidney cells, with the effects being reversible with the removal of the growth factor from the medium. Full transformation activity requires the presence of TGF-b [lo]. A second important characteristic of TGF-a is its ability to bind to EGF receptors. After TGF-a b!nds to the 170-kDa glycoprotein EGF receptor, it activates the intrinsic tyrosine kinase, then receptor autophosporylation, internalization and downregulation occurs [28]. Downward cf al. jl3] have demonstrated the homology between the EGF receptor and the oncogene protein v-erb-B. This similarity between the receptor which recognizes TGF-a! and an oncogene protein suggests that an abnormal level of the growth factor receptor may lead a signal to proliferate. Ozanne er al. [35] have substantiated this possibility by showing that human squamous carcinomas have 2- to IO-fold elevated levels of EGF receptors compared to normal skin and that the epidermoid carcinoma cell line A431 has elevated mRNA for EGF receptor and an amplification of the gene for the EGF receptor. cDNA of human TGF-a has been cloned and the nucleic acid and the peptide sequence of TGF-a have begun to be characterized. Demyck [12] reports that the gene for human TGF-cr is 70-100 kb and the TGF-a mRNA is ~4.5 kb. The human TGF-ar gene has been localized by in situ hybridization to human chromosome 2~13 [5, 451. Hayward et al. [ 181and Murray et al. [33] have found restriction fragment length polymorphism for the human TGF-a gene, with the restriction
endonucleases BamH I, Rsa I and Taq I recognizing the polymorphisms. The propeptide produced from translation of human TGF-a! mRNA is 160 amino acids in length. The TGF-cw propeptide is glycosylaled and secreted by the cells which produce them and is processed like other secreted polypeptides, with amino acid positions 38 and 88 in the propeptide acting as sites for further processing [24]. The mature peptide has a final length of 50 amino acids and 30-40 % amino acid homology to EGF. There are 6 cysteine residues, with the 3 resultant disulfide bond positions conserved between TGF-cr: and EGF. The secondary structure of TGF-a: in solution has been studied by Montelione et al. [31] using NMR spectroscopy. In the mature TGF-a peptide, the authors found 2 antiparallel beta-sheets. If one takes into account the structural constraints required by the 3 disultide bonds, along with the anti-parallel sheets, the secondary structure of human TGF-cu is similar to the secondary structure of human EGF. However, TGF-a has much faster amide proton/ deu;,ron exchange than EGF, suggesting that the structural dynamics of TGF-ar differs from that of EGF. Physiologically, TGF-ol is functionally analogous to EGF. Like EGF, subcutaneous TGF-a! administration induces a dose-response-related acceleration of eyelid opening and incisor eruption in newborn mice [43]. In addition, TGF-a! administration retards hair growth and body weight increase in the newborn mice. TGF-(w also has been found to be a potent in vivo angiogenic factor, more potent than EGF, with the hamster cheek pouch bioassay, which measures the ability of a substance for angiogenic activity [39]. Since one major characteristic of TGF-m is the ability of the peptide to transform cells, TGF-a! has been investigated for its role in human cancers. Sherwin et al. [40] analyzed the urine of 22 patients with disseminated cancer (5 with breast cancer, 5 with lung cancer, 5 with colon cancer, 4 with malignant melanomas, 2 with sarcomas and 1 with ovarian cancer). They found that 18 of 22 cancer patients had a high molecular weight peptide which had TGF-a-like activity while only 5 of 22 controls had the same activity in the urine. High performance liquid chromotography (HPLC) analysis of urines of cancer patients by Kimball ef al. [22] showed that the cancer patient urines contained 5 separate peaks of TGF-a-like activity, 2 of which were more elevated in the cancer patients than in the controls. More recently, Arteaga et al. [l, 161 mea-
Transforming growth factor* sured the pleural or ascitic effusions from 130 patients with different types of cancers. They found that 42 % of the effusions contained TGF-a (compared to 18 % of noncancer effusions), with statistical correlations between the presence of TGF-(Y and the number of metastic sites and the presence of TGF-cr and survival. Thus, in the preliminary studies, it appears that the presence of TGF-cu is associated with human cancers. Below, we review the findings regarding TGF-a! in individual human cancers. TGFcl in liver cancer In human hepatic cancer, TGF-a! in the urine is associated with the presence of tumor. Rat hepatic carcinoma cells appear to produce large quantities of TGF-a. Additionally, TGF-ar levels are increased with liver regeneration, suggesting that this growth factor is associated with active cellular division of hepatic cells. Human hepatoma We studied 31 patients with hepatocellular carcinoma, 15 with probable hepatic carcinoma, 4 with metastatic liver cancer and 33 age- and sexmatched controls [47] and found that TGF-(r in the urine is significantly higher in the patients with cancer. In the hepatocellular cancer patients, was concentration urinary TGF-(w 2 1.89 + 21 .OOpg/g creatinine. In the non-cancerous controls, the mean TGF-cr concentration was 4.94 f 2.77 pg/g creatinine (P< 0.05). There was no difference in the urinary EGF concentrations between the two groups. In iiiis study, we also compared the sensitivity of urinary TGF-a to serum a-fetoprotein to detfect hepatocellular carcinoma. Alpha-fetoprotein alone detected 58.7 % of the carcinomas. TGF-a was more sensitive as a marker and detected 7 1.7 % of the cancers. The combined sensitivity of using both tests for detecting liver cancer was 93.5 %. Thus, TGF-ar could effectively serve as another marker to detect and monitor progress of patients with hepatocellular carcinomas. Rat hepatocellular carcinoma cells Rat hepatoma cells produce and secrete the mature TGF-a! peptide. The rat hepatocellular cell line JMl was originally established from a liver tumor in a Fisher 344 rat given the carcinogen diethylnitrosamine. Northern blotting of RNA from JMl cells show that the cells produce a 4.5 kb TGF-a! mRNA [24]. Studies using HPLC, radioimmunoassay and Western blotting of the
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JMl medium reveal that TGF-a! and its precursor peptide are produced in significant quantities. This study confirms in vitro that hepatocellular cancer cells can produce large amounts of TGF-a and underlines the importance of our studies of urinary TGF-ar excretion in humans with hepatocellular carcinoma [47]. Liver regeneration Liver regeneration represents another model system for studying factors regulating rapid cellular proliferation. Mead and Faust0 [30] performed partial hepatectomies in rats to examine the factors involved in liver regeneration. They found that TGF-(r expression increased after partial hepatectomy. The mRNA for TGF-a! was undetectable in the liver until 12 h after surgery, and then the TGF-c~ mRNA concentrations peaked at 24 h after surgery, coincident with the peak of cellular division. The TGF-a mRNA levels in the hepatocytes increased 9-fold between the beginning of detectable TGF-a levels and peak levels. In primary liver cell cultures, the addition of TGF-a! was associated with a 13-fold elevation of DNA synthesis. Moreover, in the cells undergoing rapid DNA synthesis, the level of TGF-a! mRNA increased. In this model system of rapid cellular proliferation, TGF-(r acts as an autocrine or paracrine factor. This study may help to explain the self-activating nature of the elevations of TGF-a! production in human hepatomas, once the initiation events for cancer growth occur. TGFcl and gastrointestinal cancer TGF-cr is produced by both normal and cancerous gastrointestinal (GI) tissues. The level of TGF-a! production in cancers and the role of TGF-a! in GI cancers are currently under active investigation. Gastrointestinal tissues and tumors In gastric tumors, TGF-(U production may be elevated. In a series of 38 gastrectomies 121,26 of which were for gastric cancers, 63 separate gastric biopsy specimens were obtained. TGF-(U mRNA was present in both cancerous and noncancerous specimens. In 16 paired specimens, where both cancerous and noncancerous tissues were obtained from the same patient, TGF-a mRNA was found to be present in higher quantities in the cancerous regions. In a very small study 1261, Northern blot analysis was used to investigate biopsies of human gastrointestinal tract from the esophagus to the colon. The investigators de-
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tected the presence of TGF-a! mRNA, after p01y A+ purification, from all parts of the normal intestinal tract, with the highest abundance in the duodenum. In 4 patients with primary colon cancers, TGF-a mRNA was also detected, albeit in quantities which were similar to that observed in normal tissues. In two patients with gastric carcinoma, they also did not detect a difference in the mRNA levels between the normal and cancerous tissue. The conflicting data concerning TGF-a in human gastrointestinal cancers need to be resolved by studying larger series of patients before definitive statements can be made concerning the role of the growth factor as a marker for diagnosis or prognosis in gastrointestinal cancer. Cell lines Human colon cancer cell lines produce TGF-ol. Coffey er al. [S] and Hanauske et al. [ 171measured TGF-ar production by human colon cancer cell lines and found that a majority of cell lines were able to produce TGF-a. In addition, the colon cell lines produce the receptor for TGF-a [8]. Buick et ai. 161studied the transformation of rat intestinal epithelium with the oncogene H-r-as. The oncogene transformation of the ceils was associated with an overexpression of TGF-rw mRNA. This overexpression of mRNA by transformed cells is consistent with the in virro Endings of elevated TGF-cr production by the colon cancer cell lines. TGFa in breast cancer Breast cancer is one of the commonest forms of cancer in American women. The mediators of breast cancer growth other than estrogen are still not well understood. TGF-a-like activity has been detected in normal breast tissue [36] and in human breast milk [49]. An abnormal production of TGF-a may, therefore, be associated with breast cancer growth. Human breast cancer : TGFc( gene expression In understanding the role of TGF-a in human breast cancer tissue growth, two important questions are : (1) is there an association between TGF-a expression and breast cancer ? and (2) is there a relationship between TGF-a expression and the presence of estrogen receptors ? Three studies have been published recently which attempt to address these questions. Travers ef al. 1441 studied TGF-a mRNA expression of 52 malignant breast biopsies and 15 nonmalignant tumors of the breast and 4 normal breast specimens. They detected TGF-a mRNA in 38 % of the
breast cancer tissues and in 22 % of normal breast biopsies. They also studied the expression of the TGF-a! receptor, the EGF receptor, in the breast specimens and found EGF receptor mRNA expression in 42 % of 45 cancer specimens while it was detectable in 100 % of 15 benign specimens. Macias et al. [25] used EGF-binding competition as their assay for TGF-a! activity and found that 31% of 45 primary mammary carcinomas had measurable TGF-a! activity. They did not use a normal group for comparison. In a small study, Perroteau et al. [36] measured the immunoreactive TGF-a activity in breast biopsies. In their series of 22 primary breast cancer biopsies and 4 noncancerous breast biopsies, all the specimens contained TGF-rr activity by radioimmunoassay, but there was no difference in the levels of TGF-(r in the cancerous specimens compared to the 4 normals. Increased TGF-cr expression may occur in breast cancers, but the degree and the type of overexpression still needs to be determined. The relationship between the presence of estrogen receptor and TGF-(w in breast cancer is yet unresolved. Travers et al. [44] found that TGF-a! mRNA and the mRNA for the TGF-cw receptor, the EGF receptor, were more than twice as common in the breast cancers which were negative for estrogen receptors. In contrast, Macias et al. [25] and Perroteau et af. [36] found no association between the estrogen receptor content of breast tumors and TGF-a levels. Urinary excretion of TGFcr The presence of TGF-ar in the urine of patients with disseminated breast cancers has been documented by Stromberg el al. 1421. They collected 22 I of urine from 3 patients with disseminated breast cancer and by chromatography, EGF-receptor competition and TGF-ar radioimmunoassay, they demonstrated that TGF-a was present in the urine of these patients. It would be useful to extend their studies to see if TGF-cr could serve as a tumor marker for diagnostic or prognostic purposes. Breast cancer cell lines In breast cancer cell lines, TGF-ar appears to stimulate cellular growth. Using the human mammary tumor cell line ZR-75-1, Novak-Hofer et at. [34] measured the relationship between TGF-a! and the phosphorylation of S6, a ribosomal protein. They showed that TGF-cw increases cell growth in this mammal cell line and phosphorylates the S6 kinase. In addition, they
Transforming growth factor* showed that the mechanism for the TGF-cu mediated increase in cell number is probably different from the mechanism for the increase in cell number by estradiol-there was no change in the level of S6 phosphorylation with estrogen stimulated cell growth. Bjorge et al. [4] have studied the human breast cancer cell line MDA468. They found that phorbol ester and EGF stimulate the production of mRNA for TGF-a and its receptor, the EGF receptor. They concluded that this growth factor could serve as an autocrine type of a signal to the cell line. Valverius ef al. [46] studied normal tissue from breast reduction mammoplasties and transformed the normal tissues with several combinations of carcinogenic drugs. In contrast to the other studies, they found that mRNA for TGF-a was no different between the normal cells and the immortalized cells derived from the normal cells. However, their findings do not necessarily reflect the situation in vivo, where the transformation process to cancer is probably different and where breast cancer cells could behave differently from in vitro. TGF-cl in melanoma and skin diseases With the similarities between TGF-a and EGF, it would be consistent for TGF-a to have an effect on the human epidermis. TGF-a is present in normal human skin. Coffey et al. [7] found that primary cultures of human keratinocytes produced TGF-a mRNA. In addition, incubations of these cells with TGF-a increased TGF-a mRNA production, suggesting the potential for auto-induction for these cells. In normal skin biopsies, in situ hybridization and immunohistochemistry showed the presence of TGF-a mRNA and protein. Melanoma TGF-a has been isolated from the urine of patients with melanoma. Sherwin er al. [40] found high molecular weight TGF-a in the urine of patients with disseminated melanoma. Using 3 I of urine from patients with melanoma, Kim et al. [21] isolated and purified by 200 OOO-fold a low molecular weight (4545 Da) form of TGF-a from the urine of these patients. Melanoma cell lines produce TGF-a. Marquardt and Todaro [27] found that the human metastatic melanoma cell line A2058 produced TGF-a. Richmond et al. [37] found that the human malignant melanoma cell line Hs0294
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produced TGF-cu. Ellem et al. [I51 tested 5 melanoma cell lines and found that all responded to ultraviolet radiation (UVR) with production of TGF-a. They found that a 12-h exposure of normal foreskin melanocytes to UVR resulted in the cellular production of TGF-a. Thus, they hypothesized that UVR resulted in the production of TGF-a, which subsequently is involved in the autocrine induction of abnormal melanocyte response. This finding may explain the origin of abnormal melanocyte cell growth, leading to the development of melanoma. Restriction fragment length polymorphism of the human TGF-a gene found in melanoma cell lines and melanoma biopsy specimens may eventually be used to identify susceptible tissues for melanoma. Using the restriction endonuclease Taq I to analyze the DNA, Hayward et al. [19] found that a 2.7 kb fragment was associated with melanoma cell lines. A large series showing a statistical correlation for the association in human melanoma would be beneficial in this regard. Psoriasis Epidermal hyperproliferation occurs in psoriasis, a common benign disease of the epidermis. Elder et al. [14] studied TGF-a expression in biopsies of psoriatic epidermis, normal-appearing skin of patients with psoriasis and normal skin. They used scanning densitometry of Northern blots to measure the TGF-a mRNA in these skin specimens. Psoriatic TGF-a mRNA was increased 4.4-fold in total mRNA preparations and 5.7-fold in poly A+ RNA preparations when compared to normal controls. In their measurements of the TGF-a peptide obtained by acid-ethanol extraction of the cells, they found a 6-fold increase in the TGF-a protein content of the psoriatic cells. Thus, it appears that TGF-a expression is a component that is actively increased in the hyperproliferative state of psoriasis. Cell cultures TGF-a was able to stimulate oncogene protein production in fibroblasts. Cutry ef al. [9] studied the fibroblast cell line C3H lOTI/ and found that TGF-a stimulated DNA synthesis. In addition, TGF-a causes a large increase in mRNA for two oncogenes, c-fos and c-myc, suggesting that one possible mechanism for the effects of TGF-a on skin cells, in addition to auto-induction, could be the stimulation of oncogene protein production.
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lGFu in lung cancer TGF-a has been detected in the urine and plural effusions of patients with lung cancer [I, 401. BY using cell lines, several additional observations have been made concerning lung cancer. Soderdahl et al. [41] found that TGF-a is expressed in 4 of 6 nonsmall cell human lung cancer cell lines. They found that TGF-a was expressed in a squamous-cell carcinoma, 2 adenocarcinomas and one large cell carcinoma cell line. Bergh PI extended this study and found that there was an association with TGF-a! production and the ability of the lung cancer cells to produce stroma fibrosis when the cancer cells were transplanted into nude mice. It then appears that TGF-a! expression may be correlated with some of the phenotypical characteristics of some lung cancer cell lines. By the use of monoclonal antibodies to human TGF-a., Imanishi et al. [20] demonstrated that the antibodies could inhibit lung cancer cell growth. They incubated an anti-human TGF-a monoclonal antibody with two different human lung adenocarcinoma cell lines (A-549 and PC-g). The growth of the cancer cells significantly decreased with the addition of the monoclonal antibody to the culture medium. This inhibitory effect was reversible with the addition of high levels of TGF-CL The significant implication from this study is that monoclonal antibodies could serve as a potential method for the control of lung cancer proliferation. TGFu in brain cancers Ex first brain tumor to be studied for TGF-a! was human glioma. Samuels et al. [38] used polyclonal antibodies to TGF-a to study benign and malignant human gliomas. They chose this particular tumor to study because of the direct relationship between the morphology of the tumor and malignancy. They found a strong relationship between the malignant nature of the tumors and the presence of TGF-a. In gliomas, therefore, TGF-a could serve as a protein marker for the degree of malignancy. TGFu in the genital and urinary tracts For ovarian cancer, Sherwin et al. [40] found elevated levels of TGF-LYin the urine of a patient with disseminated disease. Arteaga et al. [l] found e!evated levels of TGF-a in the ascitic effusions of 13 of 31 patients with ovarian cancer. Detailed reports concerning TGF-a in bladder cancer have yet to he published. Yura et al. [48], however,
studied the potential tumor enhancing effects of rat urine on rat urinary bladder cell growth. They measured omithine decarboxylase inducing activity, which is a characteristic of rodent skin tumor promoters, in the target rat bladder cancer cell line and found that TGF-a: and EGF have dose-response-related ability to induce this enzyme activity. Thus, TGF-a! as a component of the excretion products of urine could cause events leading to cancer formation in rats. Potential diagnostic and therapeutic use of TGF+x in cancer A summary of the relationship of TGF-(U to human cancer presented in this review is shown in Table I. Given these findings, one can speculate on the potential use of TGF-a! in cancer diagnosis and therapeutics. The situations in which abnormalities of TGF-a production have been noted could be used advantageously to serve as cancer markers. For example, TGF-a present in the urine of patients with hepatoma could be used as a measure of tumor load. Another example is the use of TGF-a in pleural or ascitic effusions to measure the extent of the disease. In addition, measurements of TGF-ar could be used in conjunction with other known markers of tumors, like a-fetoprotein and estrogen receptors, to increase the diagnostic techniques available to patients. In our laboratory (Yeh et al., submitted), TGF-a could be readily measured in small quantities of saliva. Therefore, in addition to using urine and bodily effusions to measure TGF-a! levels, saliva could be used to quantitate TGF-a! levels in cancer patients. Therapeutically, an understanding of the role of TGF-a could lead to treatment of human cancers. As presented in Table II, there are a number of mechanisms in which TGF-a could influence the growth of cancerous cells. Each of the potential sites of TGF-(r action could serve as a site to inhibit cancer growth, if abnormal TGF-(u activity were the cause of the cancer. One example of the use of this type of intervention is the work performed by Imanishi et al. [20] where monoclonal antibodies to TGF-cu could be administered to block carcinoma growth. Another possible method of treating cancer patients, given Samuel et al.3 [38] finding of the relationship of elevated TGF-a! levels in malignant human brain cancers, is that TGF-a could be used as a marker for targeted chemotherapy, with TGF-a serving as a marker for high concentrations of malignant cells.
Transforming
growth factora
and human cancer
657
Table I. TGF-a in human cancers. Human cancer Liver Intestinal
Breast
Findings Elevated TGF-a in urine of hepatoma
tract
Elevated Elevated pancreas Elevated
Ref:
patients
TGF-a mRNA in gastric cancers TGF-a in effusions of patients with colon, stomach and cancers TGF-a in the urine of colon cancer patients
Elevated TGF-a mRNA
in primary breast cancers
TGF-a in urine of breast cancer patients Elevated TGF-a in effusions of patients with breast cancer Skin
TGF-a
Lung
Elevated TGF-a
in effusions of patients with lung cancer
Brain Ovary
Elevated Elevated Elevated Elevated
in urine of lung cancer patients associated with malignant forms of gliomas in urine of patient with ovarian cancer in effusions of patients with ovarian cancer
in urine of melanoma
TGF-a TGF-a TGF-a TGF-a
Table II. Potential mechanisms in human cancers.
patients
for the role of TGF-a
Mechanism Abnormal elevation of TGF-a protein levels Loss of control of TGF-a gene regulation Abnormal amplification of TGF-a gene number Change in paracrine or autocrine control of TGF-a production Change in receptor number of receptor gene number for TGF-a Change in post-receotor effects of TGF-a Change in synergistic effects with other hormones, growth factors or oncogenes
Table III. Inducers of TGF-a Inducer Addition of TGF-a Gonadotropins Estrogens Progestins EGF Phorbol ester Ultraviolet radiation Regenerative state Oncogene transformation
Yeh et al.
[47l
Bennett et al.
[21
Arteaga et al. Sherwin et al. Travers et a!. Stromberg et al. Arteaga et al. Kim et al.
111
WI WI [42]
PI [211
Arteaga et al. Sherwin et al.
WI
Samuels et al.
[381
Sherwin et al. Arteaga et al.
WI
PI
111
!f e!evated TGF-a is a major factor in cancer cell growth, the knowledge concerning inducers of TGF-a! production presented in Table III couiJ be used to help prevent malignant growth. If the factors which lead to induction of abnormal TGF-a production in organs susceptible to cancer could be blocked, one may be able to hinder the cellular proliferative steps which could lead to malignancy. Blocking TGF-a! production, one after ultraviolet radiation could hypothesize, exposure in patients who are susceptibie to melanoma or blocking TGF-a! expression after estrogen and/or progesterone exposure in patients who are susceptible to breast cancer might reduce the susceptible patient’s risk of cancer formation or growth.
production.
Example Primary skin cultures Rat ovary Breast cancer cell line Breast cancer cell line Breast cancer cell line Breast cancer cell line Melanoma cell ‘line Liver regeneration Rat intestinal cells
ReJ Coffey ef a/. Kudlow et al. Perroteau et al. Murphy 8s Dotzlaw Bjorge et al. Bjorge et al. Ellem et al. Mead & Faust0 Buick et al.
171 1231 I361 ;:;I t41
WI
[301 PI
J. Yeh and Y.-C. Yeh
Conclusion In the decade since the first isolation and characterization of TGF-cu, significant steps have been made in elucidating the presence and potential roles of this growth factor in human cancers. Though detailed studies need to be performed, TGF-(w is already promising as a marker for several human cancers.
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