Cell-autonomous immune gene expression is repressed in pulmonary neuroendocrine cells and small cell lung cancer.

Small cell lung cancer (SCLC) is classified as a high-grade neuroendocrine (NE) tumor, but a subset of SCLC has been termed "variant" due to the loss of NE characteristics. In this study, we computed NE scores for patient-derived SCLC cell lines and xenografts, as well as human tumors. We aligned NE properties with transcription factor-defined molecular subtypes. Then we investigated the different immune phenotypes associated with high and low NE scores. We found repression of immune response genes as a shared feature between classic SCLC and pulmonary neuroendocrine cells of the healthy lung. With loss of NE fate, variant SCLC tumors regain cell-autonomous immune gene expression and exhibit higher tumor-immune interactions. Pan-cancer analysis revealed this NE lineage-specific immune phenotype in other cancers. Additionally, we observed MHC I re-expression in SCLC upon development of chemoresistance. These findings may help guide the design of treatment regimens in SCLC.

Small cell lung cancer tumors and preclinical models display heterogeneity of neuroendocrine phenotypes.

BACKGROUND: Small cell lung cancer (SCLC) is a deadly, high grade neuroendocrine (NE) tumor without recognized morphologic heterogeneity. However, over 30 years ago we described a SCLC subtype with "variant" morphology which did not express some NE markers and exhibited more aggressive growth. METHODS: To quantitate NE properties of SCLCs, we developed a 50-gene expression-based NE score that could be applied to human SCLC tumors and cell lines, and genetically engineered mouse (GEM) models. We identified high and low NE subtypes of SCLC in all of our sample types, and characterized their properties. RESULTS: We found that 16% of human SCLC tumors and 10% of SCLC cell lines were of the low NE subtype, as well as cell lines from the GEM model. High NE SCLC lines grew as non-adherent floating aggregates or spheroids while Low NE lines had morphologic features of the variant subtype and grew as loosely attached cells. While the high NE subtype expressed one of the NE lineage master transcription factors ASCL1 or NEUROD1, together with NKX2-1, the entire range of NE markers, and lacked expression of the neuronal and NE repressor REST, the low NE subtype had lost expression of most NE markers, ASCL1, NEUROD1 and NKX2-1 and expressed REST. The low NE subtype had undergone epithelial mesenchymal transition (EMT) and had activated the Notch, Hippo and TGFß pathways and MYC oncogene . Importantly, the high and low NE group of SCLC lines had similar gene expression profiles as their SCLC tumor counterparts. CONCLUSIONS: SCLC tumors and cell lines can exhibit distinct inter-tumor heterogeneity with respect to expression of NE features. Loss of NE expression results in major alterations in morphology, growth characteristics, and molecular properties. These findings have major clinical implications as the two subtypes are predicted to have very different responses to targeted therapies.

A comprehensively characterized cell line panel highly representative of clinical ovarian high-grade serous carcinomas.

Recent literature suggests that most widely used ovarian cancer (OVCA) cell models do not recapitulate the molecular features of clinical tumors. To address this limitation, we generated 18 cell lines and 3 corresponding patient-derived xenografts predominantly from high-grade serous carcinoma (HGSOC) peritoneal effusions. Comprehensive genomic characterization and comparison of each model to its parental tumor demonstrated a high degree of molecular similarity. Our characterization included whole exome-sequencing and copy number profiling for cell lines, xenografts, and matched non-malignant tissues, and DNA methylation, gene expression, and spectral karyotyping for a subset of specimens. Compared to the Cancer Genome Atlas (TCGA), our models more closely resembled HGSOC than any other tumor type, justifying their validity as OVCA models. Our meticulously characterized models provide a crucial resource for the OVCA research community that will advance translational findings and ultimately lead to clinical applications.

Phenotypic variation in a Chinese family with 46,XY and 46,XX 17α-hydroxylase deficiency.

BACKGROUND: 17α-hydroxylase deficiency is a rare autosomal recessive disorder characterized by sexual infantilism, amenorrhea, hypertension and hypokalemia, which is caused by mutations in the CYP17A1 gene. To date, more than 50 mutations in this gene have been described. METHODS: The clinical features and biochemical data of a pair of 46,XY and 46,XX Chinese siblings with 17α-hydroxylase deficiency from Singapore were studied. Direct DNA sequence analysis of the CYP17A1 gene was performed. RESULTS: There was significant phenotypic variation between the siblings. The proband (46,XY) presented classically with sexual infantilism, amenorrhea and hypertension. The younger sibling (46,XX) also presented with amenorrhea, but she had breast development and absence of hypokalemic hypertension. The same compound heterozygous mutations in CYP17A1 gene were identified in both patients. A missense mutation (P409R) was detected in exon 7, and a 9-bp deletion (D487-S488-F489del) was detected in exon 8. CONCLUSION: We confirmed the diagnosis of 17α-hydroxylase deficiency in these two patients. Both P409R and D487-S488-F489del have been described previously and are widely propagated in the Chinese population in East and Southeast Asia. We propose that the phenotypic expression of affected individuals with 17α-hydroxylase deficiency is karyotype-dependent, with individuals having the 46,XX karyotype having less pronounced clinical symptoms.

Dihydrotestosterone synthesis bypasses testosterone to drive castration-resistant prostate cancer.

In the majority of cases, advanced prostate cancer responds initially to androgen deprivation therapy by depletion of gonadal testosterone. The response is usually transient, and metastatic tumors almost invariably eventually progress as castration-resistant prostate cancer (CRPC). The development of CRPC is dependent upon the intratumoral generation of the potent androgen, dihydrotestosterone (DHT), from adrenal precursor steroids. Progression to CRPC is accompanied by increased expression of steroid-5α-reductase isoenzyme-1 (SRD5A1) over SRD5A2, which is otherwise the dominant isoenzyme expressed in the prostate. DHT synthesis in CRPC is widely assumed to require 5α-reduction of testosterone as the obligate precursor, and the increased expression of SRD5A1 is thought to reflect its role in converting testosterone to DHT. Here, we show that the dominant route of DHT synthesis in CRPC bypasses testosterone, and instead requires 5α-reduction of androstenedione by SRD5A1 to 5α-androstanedione, which is then converted to DHT. This alternative pathway is operational and dominant in both human CRPC cell lines and fresh tissue obtained from human tumor metastases. Moreover, CRPC growth in mouse xenograft models is dependent upon this pathway, as well as expression of SRD5A1. These findings reframe the fundamental metabolic pathway that drives CRPC progression, and shed light on the development of new therapeutic strategies.

Combined 17α-hydroxylase/17,20-lyase deficiency due to p.R96W mutation in the CYP17 gene in a Brazilian patient / Deficiência combinada de 17α-hidroxilase/17,20 liase devido à mutação p.R96W no gene CYP17 em um paciente brasileiro

Congenital adrenal hyperplasia (CAH) resulting from 17α-hydroxylase/17,20-lyase deficiency is a rare autosomal recessive disease and the second most common form of CAH in Brazil. We describe the case of a Brazilian patient with CYP17 deficiency (17α-hydroxylase/17,20-lyase deficiency) caused by a homozygous p.R96W mutation on exon 1 of the CYP17 gene, an unusual genotype in Brazilian patients with this form of CAH. The patient, raised as a normal female, sought medical care for lack of pubertal signs and primary amenorrhea at the age of 16 years. At evaluation, the presence of a 46,XY karyotype, hypertension and hypokalemia were observed. We emphasize the recognition of CYP17 deficiency in the differential diagnosis of cases of hypergonadotrophic hypogonadism and hypertension in young patients who need specific treatment for both situations.

A hiperplasia adrenal congênita (HAC), em razão da deficiência de 17α-hidroxilase/17,20-liase, é uma doença autossômica recessiva rara e a segunda causa mais comum de HAC no Brasil. Descrevemos o caso de um paciente brasileiro portador da deficiência 17α-hidroxilase/17,20- liase (CYP17) em homozigose para a mutação p.R96W no éxon 1 do gene da CYP17A1, uma mutação incomum entre os casos brasileiros descritos com essa forma de HAC. Esse paciente, criado como um indivíduo normal do sexo feminino, procurou atendimento por ausência de sinais puberais e amenorreia primária aos 16 anos de idade. Durante a avaliação, constataram-se um cariótipo 46,XY e a presença de hipertensão e hipocalemia. Enfatizamos o reconhecimento da deficiência da CYP17 dentre os possíveis diagnósticos em um paciente jovem com hipogonadismo hipergonadotrófico e hipertensão, os quais necessitam de tratamento particularizado para ambas as situações.

3beta-hydroxysteroid dehydrogenase is a possible pharmacological target in the treatment of castration-resistant prostate cancer.

Prostate cancer usually responds to androgen deprivation therapy, although the response in metastatic disease is almost always transient and tumors eventually progress as castration-resistant prostate cancer (CRPC). CRPC continues to be driven by testosterone or dihydrotestosterone from intratumoral metabolism of 19-carbon adrenal steroids from circulation, and/or de novo intratumoral steroidogenesis. Both mechanisms require 3beta-hydroxysteroid dehydrogenase (3betaHSD) metabolism of Delta(5)-steroids, including dehydroepiandrosterone (DHEA) and Delta(5)-androstenediol (A5diol), to testosterone. In contrast, reports that DHEA and A5diol directly activate the androgen receptor (AR) suggest that 3betaHSD metabolism is not required and that 3betaHSD inhibitors would be ineffective in the treatment of CRPC. We hypothesized that activation of AR in prostate cancer by DHEA and A5diol requires their conversion via 3betaHSD to androstenedione and testosterone, respectively. Here, we show that DHEA and A5diol induce AR chromatin occupancy and AR-regulated genes. Furthermore, we show that Delta(5)-androgens undergo 3beta-dehydrogenation in prostate cancer and that induction of AR nuclear translocation, AR chromatin occupancy, transcription of PSA, TMPRSS2, and FKBP5, as well as cell proliferation by DHEA and A5diol, are all blocked by inhibitors of 3betaHSD. These findings demonstrate that DHEA and A5diol must be metabolized by 3betaHSD to activate AR in these models of CRPC. Furthermore, this work suggests that 3betaHSD may be exploited as a pharmacologic target in the treatment of CRPC.

Combined 17α-hydroxylase/17,20-lyase deficiency due to p.R96W mutation in the CYP17 gene in a Brazilian patient.

Congenital adrenal hyperplasia (CAH) resulting from 17α-hydroxylase/17,20-lyase deficiency is a rare autosomal recessive disease and the second most common form of CAH in Brazil. We describe the case of a Brazilian patient with CYP17 deficiency (17α-hydroxylase/17,20-lyase deficiency) caused by a homozygous p.R96W mutation on exon 1 of the CYP17 gene, an unusual genotype in Brazilian patients with this form of CAH. The patient, raised as a normal female, sought medical care for lack of pubertal signs and primary amenorrhea at the age of 16 years. At evaluation, the presence of a 46,XY karyotype, hypertension and hypokalemia were observed. We emphasize the recognition of CYP17 deficiency in the differential diagnosis of cases of hypergonadotrophic hypogonadism and hypertension in young patients who need specific treatment for both situations.

Biochemical factors governing the steady-state estrone/estradiol ratios catalyzed by human 17beta-hydroxysteroid dehydrogenases types 1 and 2 in HEK-293 cells.

Human 17beta-hydroxysteroid dehydrogenase types 1 and 2 (17betaHSD1 and 17betaHSD2) regulate estrogen potency by catalyzing the interconversion of estrone (E1) and estradiol (E2) using nicotinamide adenine dinucleotide (phosphate) cofactors NAD(P)(H). In intact cells, 17betaHSD1 and 17betaHSD2 establish pseudo-equilibria favoring E1 reduction or E2 oxidation, respectively. The vulnerability of these equilibrium steroid distributions to mutations and to altered intracellular cofactor abundance and redox state, however, is not known. We demonstrate that the equilibrium E2/E1 ratio achieved by 17betaHSD1 in intact HEK-293 cell lines is progressively reduced from 94:6 to 10:90 after mutagenesis of R38, which interacts with the 2'-phosphate of NADP(H), and by glucose deprivation, which lowers the NADPH/NADP(+) ratio. The shift to E2 oxidation parallels changes in apparent K(m) values for purified 17betaHSD1 proteins to favor NAD(H) over NADP(H). In contrast, mutagenesis of E116 (corresponding to R38 in 17betaHSD1) and changes in intracellular cofactor ratios do not alter the greater than 90:10 E1/E2 ratio catalyzed by 17betaHSD2, and these mutations lower the apparent K(m) of recombinant 17betaHSD2 for NADP(H) only less than 3-fold. We conclude that the equilibrium E1/E2 ratio maintained by human 17betaHSD1 in intact cells is governed by NADPH saturation, which is strongly dependent on both R38 and high intracellular NADPH/NADP(+) ratios. In contrast, the preference of 17betaHSD2 for E2 oxidation strongly resists alteration by genetic and metabolic manipulations. These findings suggest that additional structural features, beyond the lack of a specific arginine residue, disfavor NADPH binding and thus support E2 oxidation by 17betaHSD2 in intact cells.

Puberty in a case with novel 17-hydroxylase mutation and the putative role of estrogen in development of pubic hair.

OBJECTIVE: 17-Hydroxylase/17,20-lyase deficiency (17OHD) results from mutations in the CYP17A1 gene, leading to failure to synthesize cortisol, adrenal androgens, and gonadal steroids. Adrenarche is a consequence of the increased production of adrenal androgens. Here, we report a case carrying novel R239Q mutation causing complete functional loss of CYP17A1, and thus absence of adrenal and gonadal sex hormone production. The patient has had unexpected pubic hair development and insufficient breast development with estrogen replacement therapy. Possible mechanisms leading to pubic hair development and breast underdevelopment are discussed. PATIENT AND METHODS: A 15-year-old female born to consanguineous parents presented with the lack of full breast development and irregular menses after the age of 14 years. She had Tanner III breast development on one side, Tanner I on the other side and Tanner I pubic hair and, no axillary hair development. The serum levels of FSH, LH, and progesterone were high and, estradiol was low. The measurement of basal and ACTH-stimulated steroids was consistent with the diagnosis of 17OHD. Genetic analysis revealed novel homozygous mutation R239Q in CYP17A1 gene. Therapy with hydrocortisone was initiated and followed by the addition of conjugated estrogen. Her breast development did not improve considerably, however, pubic hair development started after estrogen treatment in spite of undetectable serum levels of androgens. CONCLUSION: This case study suggests that estrogen exerts a permissive effect on pubic hair development in girls, even in the presence of very low-circulating androgens, and impaired breast development might be due to estrogen/progesterone imbalance in breast tissue.

Cofactors, redox state, and directional preferences of hydroxysteroid dehydrogenases.

The hydroxysteroid dehydrogenases (HSDs) interconvert pairs of weak and potent steroids, thus serving as key enzymes in the regulation of intracellular hormone potency. These enzymes may appear to drive unidirectional steroid flux in intact cells but actually catalyze bi-directional metabolism that achieve pseudo-equilibria with strong directional preferences. Even small shifts in the magnitude of these pseudo-equilibria can profoundly change steroid potency and thus contribute to disease. Consequently, we are studying the structural and biochemical principles that govern these directional preferences and the resilience of these pseudo-equilibria in intact cells. HSD directional preferences in intact cells are governed largely by relative affinities for nicotinamide cofactors [NAD(P)(H)] and existing cofactor gradients. We can attenuate the directional preferences for human 17betaHSD type 1 and rat AKR1C9 in intact cells by either diminishing the NADPH/NADP(+) gradient or by mutating the arginine residues that form salt bridges with the 2'-phosphate of NADP(H) (R38 and R276, respectively).

Two novel mutations found in a patient with 17alpha-hydroxylase enzyme deficiency.

CONTEXT: Congenital adrenal hyperplasia resulting from 17alpha-hydroxylase deficiency (17OHD) is a rare disorder associated with hypertension. SUBJECT AND METHODS: We describe a phenotypically and hormonally affected female patient with 17OHD. DNA sequencing of her CYP17 gene revealed a maternal heterozygous mutation in exon 2 (R125Q) and a paternal heterozygous mutation in exon 8 (R416H). These are novel mutations in the CYP17 gene that completely eliminate enzyme activity. CONCLUSION: Identification of novel mutations in the CYP17 gene is vital in understanding the molecular mechanisms of its deficiency and in providing additional information about the structure and enzymatic functions of P450c17.

Arginine 276 controls the directional preference of AKR1C9 (rat liver 3alpha-hydroxysteroid dehydrogenase) in human embryonic kidney 293 cells.

Rat liver AKR1C9 is the best-studied 3alpha-hydroxysteroid dehydrogenase (3alphaHSD) of the aldo-keto reductase superfamily. The physiologic function of AKR1C9 is to catalyze the reduction of 5alpha-androstane-17beta-ol-3-one (dihydrotestosterone) to 5alpha-androstane-3alpha,17beta-diol (androstanediol) rather than the reverse reaction, and all of the known AKR1C enzymes with 3alphaHSD activity also preferentially catalyze dihydrotestosterone reduction in intact cells. Because the utilization of pyridine-nucleotide cofactors NAD(P)(H) primarily governs the directional preference of HSD enzymes in intact cells, and because R276 participates in NADP(H) binding, we hypothesized that mutation of R276 would alter directional preference in intact cells. To test this model, we constructed stable lines of human embryonic kidney 293 cells expressing wild-type AKR1C9 and mutations R276M, R276G, and R276E. Mutations R276M and R276G retained reductive preference with slightly reduced magnitude compared with wild-type AKR1C9. NADPH depletion by glucose deprivation minimally altered the equilibrium steroid distribution for wild-type AKR1C9 but further reduced the reductive preference of mutations R276M and R276G. Mutation R276E, in contrast, showed an oxidative preference under all conditions. The intrinsic rates of the reductive and oxidative reactions for all four enzymes were similar at the functional equilibrium states. We conclude the R276 maximizes the reductive preference of AKR1C9 in intact cells and maintains this strong preference despite NADPH depletion; mutation R276E reverses the directional preference.