Leucine can modulate skeletal muscle metabolism by enhancing protein synthesis and decreasing proteolysis. In this study, we investigated the effects of leucine on the ubiquitin–proteasome system in skeletal muscle of pregnant tumour-bearing rats fed a leucine-rich diet. Pregnant Wistar rats were distributed into three groups that were fed a semi-purified control diet (C, control; W, Walker tumour-bearing; P, pair-fed) and three other groups of pregnant rats fed a semi-purified leucine-rich diet (L, leucine; WL, Walker tumour-bearing; PL, pair-fed). The tumour-bearing rats were injected subcutaneously with a suspension of Walker 256 tumour cells. Protein synthesis and degradation were measured in gastrocnemius muscle; the total protein content and tissue chymotrypsin-like and alkaline phosphatase enzyme activities were also determined. Muscle protein extracts were run on SDS-PAGE to assess the expression of the myosin heavy chain (MHC), 20S α proteasome subunit, 19S MSSI ATPase regulator subunit and 11S α subunit. Although tumour growth decreased the incorporation of [3H]-Phe, the concomitant feeding of a leucine-rich diet increased the rate of protein synthesis. Muscle proteolysis in both tumour-bearing groups was increased more than in the respective control groups. Conversely, the leucine-rich diet caused less protein breakdown in the WL group than in the W group. Only the W group showed a significant reduction (71%) in the myosin content. In WL rats, the 20S proteasome content (32 kDa band) was reduced, while the expression of the 19S subunit was 3-fold less than in the W group and the 11S proteasome subunit reduced, to around 32% less than in the W group. These findings clearly indicate that leucine can stimulate protein synthesis and inhibit protein breakdown in pregnant rats, probably by modulating the activation of the ubiquitin–proteasome system during tumour growth.
G Ventrucci, M A R Mello and M C C Gomes-Marcondes
S Latteyer, S Christoph, S Theurer, G S Hönes, K W Schmid, D Führer and L C Moeller
Thyroid hormones are important for physiology and homeostasis. In addition to nuclear thyroid hormone receptors, the plasma membrane protein integrin αvβ3 has been recognized as a receptor for both thyroxine (T4) and triiodothyronine (T3). Here, we studied whether thyroid hormone promotes growth of murine lung cancer via αvβ3 in vivo. Murine Lewis lung carcinoma cells (3LL), stably transfected with luciferase, were injected into mouse lungs. Tumor growth in untreated mice was compared to hypothyroid mice and hypothyroid mice treated with T3 or T4 with or without the αvβ3 inhibitor 3,5,3′,5′-tetraiodothyroacetic acid (Tetrac). Tumor progression was determined by serial in vivo imaging of bioluminescence emitted from the tumor. Tumor weight was recorded at the end of the experiment. Neoangiogenesis was determined by immunohistochemistry for CD31. Tumor growth was reduced in hypothyroidism and increased by T4 treatment. Strikingly, only T4 but not T3 treatment promoted tumor growth. This T4 effect was abrogated by the αvβ3 inhibitor Tetrac. Tumor weight and neoangiogenesis were also significantly increased only in T4-treated mice. The T4 effect on tumor weight and neoangiogenesis was abolished by Tetrac. In vitro, T4 did not stimulate 3LL cell proliferation or signaling pathway activation. We conclude that T4 promotes lung cancer growth in this orthotopic mouse model. The tumor-promoting effect is mediated via the plasma membrane integrin αvβ3 and increased neoangiogenesis rather than direct stimulation of 3LL cells. These data suggest that such effects of levothyroxine may need to be considered in cancer patients on T4 substitution.
Daphne R Pringle, Zhirong Yin, Audrey A Lee, Parmeet K Manchanda, Lianbo Yu, Alfred F Parlow, David Jarjoura, Krista M D La Perle and Lawrence S Kirschner
Thyroid cancer is the most common endocrine malignancy in the population, and the incidence of this cancer is increasing at a rapid rate. Although genetic analysis of papillary thyroid cancer (PTC) has identified mutations in a large percentage of patients, the genetic basis of follicular thyroid cancer (FTC) is less certain. Thyroid cancer, including both PTC and FTC, has been observed in patients with the inherited tumor predisposition Carney complex, caused by mutations in PRKAR1A. In order to investigate the role of loss of PRKAR1A in thyroid cancer, we generated a tissue-specific knockout of Prkar1a in the thyroid. We report that the resulting mice are hyperthyroid and developed follicular thyroid neoplasms by 1 year of age, including FTC in over 40% of animals. These thyroid tumors showed a signature of pathway activation different from that observed in other models of thyroid cancer. In vitro cultures of the tumor cells indicated that Prkar1a-null thyrocytes exhibited growth factor independence and suggested possible new therapeutic targets. Overall, this work represents the first report of a genetic mutation known to cause human FTC that exhibits a similar phenotype when modeled in the mouse. In addition to our knowledge of the mechanisms of human follicular thyroid tumorigenesis, this model is highly reproducible and may provide a viable mechanism for the further clinical development of therapies aimed at FTC.
K-M Fung, E N S Samara, C Wong, A Metwalli, R Krlin, B Bane, C Z Liu, J T Yang, J V Pitha, D J Culkin, B P Kropp, T M Penning and Hsueh-Kung Lin
Type 2 3α-hydroxysteroid dehydrogenase (3α-HSD) is a multi-functional enzyme that possesses 3α-, 17β- and 20α-HSD, as well as prostaglandin (PG) F synthase activities and catalyzes androgen, estrogen, progestin and PG metabolism. Type 2 3α-HSD was cloned from human prostate, is a member of the aldo-keto reductase (AKR) superfamily and was named AKR1C3. In androgen target tissues such as the prostate, AKR1C3 catalyzes the conversion of Δ4-androstene-3,17-dione to testosterone, 5α-dihydrotestosterone to 5α-androstane-3α,17β-diol (3α-diol), and 3α-diol to androsterone. Thus AKR1C3 may regulate the balance of androgens and hence trans-activation of the androgen receptor in these tissues. Tissue distribution studies indicate that AKR1C3 transcripts are highly expressed in human prostate. To measure AKR1C3 protein expression and its distribution in the prostate, we raised a monoclonal antibody specifically recognizing AKR1C3. This antibody allowed us to distinguish AKR1C3 from other AKR1C family members in human tissues. Immunoblot analysis showed that this monoclonal antibody binds to one species of protein in primary cultures of prostate epithelial cells and in LNCaP prostate cancer cells. Immunohistochemistry with this antibody on human prostate detected strong nuclear immunoreactivity in normal stromal and smooth muscle cells, perineurial cells, urothelial (transitional) cells, and endothelial cells. Normal prostate epithelial cells were only faintly immunoreactive or negative. Positive immunoreactivity was demonstrated in primary prostatic adenocarcinoma in 9 of 11 cases. Variable increases in immunoreactivity for AKR1C3 was also demonstrated in non-neoplastic changes in the prostate including chronic inflammation, atrophy and urothelial (transitional) cell metaplasia. We conclude that elevated expression of AKR1C3 is highly associated with prostate carcinoma. Although the biological significance of elevated AKR1C3 in prostatic carcinoma is uncertain, AKR1C3 may be responsible for the trophic effects of androgens and/or PGs on prostatic epithelial cells.
R J Santen, W Yue, F Naftolin, G Mor and L Berstein
Substantial evidence supports the concept that estrogens cause breast cancer in animals and in women but the precise mechanism is unknown. The most commonly held theory is that estrogens stimulate proliferation of breast cells and thus statistically increase the chances for genetic mutations which could result in cancer. Another theory is that estrogen metabolism generates oxygen-free radicals and quinones which produce both stable and unstable DNA adducts. Both result in genetic mutations which accumulate and could ultimately cause cancer. A major criticism of the latter hypothesis is that breast tissue contains insufficient concentrations of estrogen for accumulation of genotoxic metabolites. Our hypothesis is that breast tissue estrogen levels, as a result of in situ synthesis, are much higher than previously thought. We and others have shown that estrogen can be made in the breast itself through conversion of androgens to estrogens, a process catalyzed by the enzyme aromatase. The levels of estrogen in the breast increase when aromatase is overexpressed. With sufficient amounts of aromatase in breast tissue, enough estradiol as substrate should be available to allow formation of substantial amounts of genotoxic metabolites. We postulate that aromatase overexpression may in this way cause breast cancer. As evidence supporting this concept, four animal models of aromatase overexpression and either breast cancer or premalignant lesions have been described. We have provided evidence that normal breast tissue can make estrogen and that certain stimulatory compounds can increase aromatase activity in the breast by nearly 10,000-fold. If our concepts are correct, it might be possible to prevent breast cancer by blocking the aromatase enzyme. Drugs are currently available to inhibit aromatase nearly completely without causing significant side-effects. Aromatase inhibitors might be more effective than antiestrogens in preventing breast cancer because of their dual role to block both initiation and promotion of breast cancer. To inhibit the initiation process, these inhibitors would reduce levels of the genotoxic metabolites of estradiol by lowering estradiol concentrations in tissue. At the same time, aromatase inhibitors would inhibit the process of tumor promotion by lowering tissue levels of estradiol and thus blocking cell proliferation. These concepts provide a strong rationale for studies of aromatase inhibitors to prevent breast cancer.
R J Santen, R X Song, Z Zhang, R Kumar, M-H Jeng, S Masamura, W Yue and L Berstein
Clinical observations suggest that human breast tumors can adapt to endocrine therapy by developing hypersensitivity to estradiol (E(2)). To understand the mechanisms responsible, we examined estrogenic stimulation of cell proliferation in a model system and provided in vitro and in vivo evidence that long-term E(2) deprivation (LTED) causes "adaptive hypersensitivity". The enhanced responses to E(2) do not involve mechanisms acting at the level of transcription of estrogen-regulated genes. We found no evidence of hypersensitivity when examining the effects of E(2) on regulation of c-myc, pS2, progesterone receptor, several estrogen receptor (ER) reporter genes, or c-myb in hypersensitive cells. Estrogen deprivation of breast cells long-term does up-regulate both the MAP kinase and phosphatidyl-inositol 3-kinase pathways. As a potential explanation for up-regulation of these signaling pathways, we found that ERalpha is 4- to 10-fold up-regulated and co-opts a classic growth factor pathway using Shc, Grb-2 and Sos. This induces rapid non-genomic effects which are enhanced in LTED cells. E(2) binds to cell membrane-associated ERalpha, physically associates with the adapter protein SHC, and induces its phosphorylation. In turn, Shc binds Grb-2 and Sos, which results in the rapid activation of MAP kinase. These non-genomic effects of E(2) produce biological effects as evidenced by Elk activation and by morphological changes in cell membranes. Further proof of the non-genomic effects of E(2) involved use of cells which selectively expressed ERalpha in the nucleus, cytosol and cell membrane. We created these COS-1 "designer cells" by transfecting ERalpha lacking a nuclear localization signal and containing a membrane localizing signal. The concept of "adaptive hypersensitivity" and the mechanisms responsible for this phenomenon have important clinical implications. Adaptive hypersensitivity would explain the superiority of aromatase inhibitors over the selective ER modulators (SERMs) for treatment of breast cancer. The development of highly potent third-generation aromatase inhibitors allows reduction of breast tissue E2 to very low levels and circumvents the enhanced sensitivity of these cells to the proliferative effects of E(2). Clinical trials in the adjuvant, neoadjuvant and advanced disease settings demonstrate the greater clinical efficacy of the aromatase inhibitors over the SERMs. More recent observations indicate that the aromatase inhibitors are superior for the prevention of breast cancer as well. These observations may be explained by the hypothesis that estrogens induce breast cancer both by stimulating cell proliferation and by their metabolism to genotoxic products. The SERMs block ER-mediated proliferation only, whereas the aromatase inhibitors exert dual effects on proliferation and genotoxic metabolite formation.
Anja Rudolph, Juan Sainz, Rebecca Hein, Michael Hoffmeister, Bernd Frank, Asta Försti, Hermann Brenner, Kari Hemminki and Jenny Chang-Claude
The mechanisms underlying the association of menopausal hormone therapy (MHT) with reduced colorectal cancer (CRC) risk are unknown and the identification of genetic modifiers may yield further insight. We explored the effect modification of MHT-associated CRC risk in postmenopausal women by 47 polymorphisms with known or putative functional relevance in 16 candidate genes related to hormone metabolism (COMT, CYP1A1, CYP1A2, CYP1B1, CYP2C9, CYP2C19, CYP3A4, CYP17A1, GSTP, and HSD17B1), transport (ABCB1), and signaling (ESR1, ESR2, SHBG, PGR, and NR1I2). A total of 685 CRC patients and 684 healthy controls from a German population-based case–control study (DACHS) were genotyped. Multiplicative statistical interaction between polymorphisms and ever MHT use as well as duration of use was assessed using multivariate logistic regression. CRC risk associated with ever MHT use as well as with duration was significantly modified by rs1202168 in the transporter gene ABCB1 (P interaction=0.04). The MHT-associated risk reduction was not significant in homozygous non-carriers (odds ratio (OR) ever use=0.84, 95% confidence interval (CI) 0.53–1.34; OR per 5 year duration=0.94, 95% CI 0.83–1.08), while homozygous carriers of the minor T allele had a 57% lower risk with ever use of MHT (95% CI 0.21–0.88) and a 22% lower risk per 5 years of MHT use (95% CI 0.62–0.97). Significant effect modification was also observed for the ESR1_rs910416 polymorphism (P interaction=0.03 for ever use and 0.07 for duration of use), whereby the decreased risk was attenuated in homozygous carriers of the minor C allele (OR ever use=0.87, 95% CI 0.48–1.60, OR per 5 year duration=0.99, 95% CI 0.83–1.18). Results of this exploratory study provide first evidence that polymorphisms in genes related to estrogen transport and signaling may modify MHT-associated CRC risk but warrant replication in an independent population.
Caterina Tiozzo, Soula Danopoulos, Maria Lavarreda-Pearce, Sheryl Baptista, Radka Varimezova, Denise Al Alam, David Warburton, Rehan Virender, Stijn De Langhe, Antonio Di Cristofano, Saverio Bellusci and Parviz Minoo
Even though the role of the tyrosine phosphatase Pten as a tumor suppressor gene has been well established in thyroid cancer, its role during thyroid development is still elusive. We therefore targeted Pten deletion in the thyroid epithelium by crossing Pten flox/flox with a newly developed Nkx2.1-cre driver line in the BALB/c and C57BL/6 genetic backgrounds. C57BL/6 homozygous Pten mutant mice died around 2 weeks of age due to tracheal and esophageal compression by a hyperplasic thyroid. By contrast, BALB/c homozygous Pten mutant mice survived up to 2 years, but with a slightly increased thyroid volume. Characterization of the thyroid glands from C57BL/6 homozygous Pten mutant mice at postnatal day 14 (PN14) showed abnormally enlarged tissue with areas of cellular hyperplasia, disruption of the normal architecture, and follicular degeneration. In addition, differing degrees of hypothyroidism, thyroxine (T4) decrease, and thyroid-stimulating hormone elevation between the strains in the mutants and the heterozygous mutant were detected at PN14. Finally, C57BL/6 heterozygous Pten mutant mice developed thyroid tumors after 2 years of age. Our results indicate that Pten has a pivotal role in thyroid development and its deletion results in thyroid tumor formation, with the timing and severity of the tumor depending on the particular genetic background.
J -D Lin, B -Y Huang and H -Y Chang
To investigate the difficulties in the diagnosis of thyroid microcarcinoma and to present the results of delaying diagnosis for these patients, we retrospectively analyzed the clinical information of 1259 thyroid carcinoma patients in one medical center. During a period of 20 years, from January 1977 to June 1997, 1259 thyroid cancer patients, including 921 papillary thyroid carcinoma patients, who received treatment and were followed-up at Chang Gung Medical Center in Linkou, Taiwan, were evaluated for inclusion in the study. Of these patients, 127 (13.2%) were diagnosed as having thyroid microcarcinoma. Forty-five patients were diagnosed as malignancy or suspicious malignancy preoperatively with ultrasonography and fine needle aspiration cytological examinations. In the analysis, the 127 thyroid microcarcinoma patients who received surgical treatment could be divided into four groups. Group I: patients with thyroid microcarcinoma with hyperthyroidism or hyperparathyroidism, in most of whom (except four patients) the thyroid microcarcinoma was found incidentally during the operation (28 cases). Group II: thyroid microcarcinoma in benign larger thyroid nodule or multinodular goiter, or thyroid microcarcinoma in coexistence with nodule goiter in one patient. The thyroid microcarcinomas in this group were found incidentally except in five patients (58 cases). Group III: thyroid microcarcinoma which could be detected as thyroid nodule preoperatively (28 cases). Group IV: thyroid microcarcinoma presented with neck lymph node metastases or distant metastases of the thyroid carcinoma (13 cases). Median follow-up period of these 127 patients was 4.7 years. During the follow-up period, two patients died, including one patient in group IV who died of skull metastasis with brain invasion. Another patient died of stroke, which was, however, not related to thyroid carcinoma. In conclusion, most thyroid microcarcinoma patients experienced rather benign clinical courses, but for patients with thyroid microcarcinoma with distant metastases, aggressive surgical treatment followed by radioactive 131I treatment is indicated.
E Fiore, T Rago, F Latrofa, M A Provenzale, P Piaggi, A Delitala, M Scutari, F Basolo, G Di Coscio, L Grasso, A Pinchera and P Vitti
The possible association between Hashimoto's thyroiditis (HT) and papillary thyroid carcinoma (PTC) is a still debated issue. We analyzed the frequency of PTC, TSH levels and thyroid autoantibodies (TAb) in 13 738 patients (9824 untreated and 3914 under l-thyroxine, l-T4). Patients with nodular-HT (n=1593) had high titer of TAb and/or hypothyroidism. Patients with nodular goiter (NG) were subdivided in TAb−NG (n=8812) with undetectable TAb and TAb+NG (n=3395) with positive TAb. Among untreated patients, those with nodular-HT showed higher frequency of PTC (9.4%) compared with both TAb−NG (6.4%; P=0.002) and TAb+NG (6.5%; P=0.009) and presented also higher serum TSH (median 1.30 vs 0.71 μU/ml, P<0.001 and 0.70 μU/ml, P<0.001 respectively). Independently of clinical diagnosis, patients with high titer of TAb showed a higher frequency of PTC (9.3%) compared to patients with low titer (6.8%, P<0.001) or negative TAb (6.3%, P<0.001) and presented also higher serum TSH (median 1.16 vs 0.75 μU/ml, P<0.001 and 0.72 μU/ml, P<0.001 respectively). PTC frequency was strongly related with serum TSH (odds ratio (OR)=1.111), slightly related with anti-thyroglobulin antibodies (OR=1.001), and unrelated with anti-thyroperoxidase antibodies. In the l-T4-treated group, when only patients with serum TSH levels below the median value (0.90 μU/ml) were considered, no significant difference in PTC frequency was found between nodular-HT, TAb−NG and TAb+NG. In conclusion, the frequency of PTC is significantly higher in nodular-HT than in NG and is associated with increased levels of serum TSH. Treatment with l-T4 reduces TSH levels and decreases the occurrence of clinically detectable PTC.