Development of the mammary gland requires both proper hormone signaling and cross talk between the stroma and epithelium. While estrogen receptor (ERα) expression in the epithelium is essential for normal gland development, the role of this receptor in the stroma is less clear. Moreover, several lines of evidence suggest that mouse phenotypes of in utero exposure to endocrine disruption act through mesenchymal ERα in the developing fetus. We utilized a Twist2-cre mouse line to knock out mesenchymal ERα. Herein, we assessed mammary gland development in the context of mesenchymal ERα deletion. We also tested the effect of in utero bisphenol A (BPA) exposure to alter the tumor susceptibility in the mouse mammary tumor virus-neu (MMTV-neu) breast cancer mouse model. Mesenchymal ERα deletion resulted in altered reproductive tract development and atypical cytology associated with estrous cycling. The mammary gland demonstrated mature epithelial extension unlike complete ERα-knockout mice, but ductal extension was delayed and reduced compared to ERα-competent mice. Using the MMTV-Neu cancer susceptibility model, ERα-intact mice exposed to BPA had reduced tumor-free survival and overall survival compared to BPA-exposed mice having mesenchymal ERα deletion. This difference is specific for BPA exposure as vehicle-treated animals had no difference in tumor development between mice expressing and not expressing mesenchymal ERα. These data demonstrate that mesenchymal ERα expression is not required for ductal extension, nor does it influence cancer risk in this mouse model but does influence the cancer incidence associated with in utero BPA exposure.

The biochemical phenotype of paragangliomas (PGLs) is highly dependent on the underlying genetic background and tumor location. PGLs at extra-adrenal locations usually do not express phenylethanolamine N-methyltransferase (PNMT), the enzyme required for epinephrine production, which was explained by the absence of glucocorticoids. PGLs with pathogenic variants (PVs) in Harvey rat sarcoma viral oncogene homolog (HRAS) can occur in or outside of the adrenal, but always synthesize epinephrine independently of the localization. Here, we characterize the signaling pathways through which PVs in HRAS influence PNMT expression. Catecholamines, cortisol, and transcriptional features of PGL tissues with known genetic background were analyzed. Genetically modified rat pheochromocytoma cells carrying PVs in Hras were generated and analyzed for regulation of Pnmt expression. Elevated epinephrine contents in PGLs with PVs in HRAS were accompanied by enrichment in mitogen-activated protein kinase (MAPK) signaling compared to PGLs with PVs in genes that activate hypoxia pathways. In vitro, Hras PVs increased Pnmt expression and epinephrine biosynthesis through increased phosphorylation of stimulatory protein 1 via MAPK signaling. Here, we provide a molecular mechanism that explains the PV-dependent epinephrine production of PGLs.

Thyroid cancer is associated with a broad range of different mutations, including RET (rearranged during transfection) fusion genes. The importance of characterizing RET fusion-positive tumors has recently increased due to the possibility of targeted treatment. The aim of this study was to identify RET fusion-positive thyroid tumors, correlate them with clinicopathological features, compare them with other mutated carcinomas, and evaluate long-term follow-up of patients. The cohort consisted of 1564 different thyroid tissue samples (including 1164 thyroid carcinoma samples) from pediatric and adult patients. Samples were analyzed for known driver mutations occurring in thyroid cancer. Negative samples were subjected to extensive RET fusion gene analyses using next-generation sequencing and real-time PCR. RET fusion genes were not detected in any low-risk neoplasm or benign thyroid tissue and were detected only in papillary thyroid carcinomas (PTCs), in 113/993 (11.4%) patients, three times more frequently in pediatric and adolescent patients (29.8%) than in adult patients (8.7%). A total of 20 types of RET fusions were identified. RET fusion-positive carcinomas were associated with aggressive tumor behavior, including high rates of lymph node (75.2%) and distant metastases (18.6%), significantly higher than in NTRK fusion, BRAF V600E and RAS-positive carcinomas. Local and distant metastases were also frequently found in patients with microcarcinomas positive for the RET fusions. ’True recurrences’ occurred rarely (2.4%) and only in adult patients. The 2-, 5-, 10-year disease-specific survival rates were 99%, 96%, and 95%, respectively. RET fusion-positive carcinomas were associated with high invasiveness and metastatic activity, but probably due to intensive treatment with low patient mortality.

Intratumoral androgen biosynthesis contributes to castration-resistant prostate cancer progression in patients treated with androgen deprivation therapy. The molecular mechanisms by which castration-resistant prostate cancer acquires the capacity for androgen biosynthesis to bypass androgen deprivation therapy are not entirely known. Here, we show that semaphorin 3C, a secreted signaling protein that is highly expressed in castration-resistant prostate cancer, can promote steroidogenesis by altering the expression profile of key steroidogenic enzymes. Semaphorin 3C not only upregulates enzymes required for androgen synthesis from dehydroepiandrosterone or de novo from cholesterol but also simultaneously downregulates enzymes involved in the androgen inactivation pathway. These changes in gene expression correlate with increased production of androgens induced by semaphorin 3C in prostate cancer model cells. Moreover, semaphorin 3C upregulates androgen synthesis in LNCaP cell-derived xenograft tumors, likely contributing to the enhanced in vivo tumor growth rate post castration. Furthermore, semaphorin 3C activates sterol regulatory element-binding protein, a transcription factor that upregulates enzymes involved in the synthesis of cholesterol, a sole precursor for de novo steroidogenesis. The ability of semaphorin 3C to promote intratumoral androgen synthesis may be a key mechanism contributing to the reactivation of the androgen receptor pathway in castration-resistant prostate cancer, conferring continued growth under androgen deprivation therapy. These findings identify semaphorin 3C as a potential therapeutic target for suppressing intratumoral steroidogenesis.

Ataxia telangiectasia and Rad3-related protein (ATR) is a critical component of the DNA damage response and a potential target in the treatment of cancers. An ATR inhibitor, BAY 1895344, was evaluated for its use in differentiated thyroid cancer (DTC) therapy. BAY 1895344 inhibited cell viability in four DTC cell lines (TPC1, K1, FTC-133, and FTC-238) in a dose-dependent manner. BAY 1895344 treatment arrested DTC cells in the G2/M phase, increased caspase-3 activity, and caused apoptosis. BAY 1895344 in combination with either sorafenib or lenvatinib showed mainly synergistic effects in four DTC cell lines. The combination of BAY 1895344 with dabrafenib plus trametinib revealed synergistic effects in K1 cells that harbor BRAFV600E. BAY 1895344 monotherapy retarded the growth of K1 and FTC-133 tumors in xenograft models. The combinations of BAY 1895344 plus lenvatinib and BAY 1895344 with dabrafenib plus trametinib were more effective than any single therapy in a K1 xenograft model. No appreciable toxicity appeared in animals treated with either a single therapy or a combination treatment. Our findings provide the rationale for the development of clinical trials of BAY 1895344 in the treatment of DTC.

Steroid cell tumors, not otherwise specified (SCT-NOS), are uncommon ovarian neoplasms accompanied by virilization symptoms due to hyperandrogenism, which are malignant in approximately one-third of the cases. Given the rarity of SCT-NOS, their molecular underpinnings have not yet been studied in depth. In this case series, we performed the first comprehensive analysis of the genetic landscape of this rare ovarian tumor. A detailed clinicopathological description of an index case is also provided. Over a 20-year period, a total of eight patients were seen at our institution. Total nucleic acids (RNA and DNA) were extracted from evaluable formalin-fixed, paraffin-embedded tumor specimens (n = 7) and subjected to TruSight Oncology 500 testing and/or exome sequencing. The results identified pathogenic variants in several hypoxia-related genes – including HIF1A, VHL, SDHB, SRC, IDH2, and FOXO4. As the first comprehensive genetic analysis of SCT-NOS, this study shows that dysregulation in the hypoxia signaling pathway is a key molecular feature of this rare tumor. Clinically, long-term follow-up with periodic measurements of androgen levels should be pursued in all cases since recurrences may occur several years after the initial diagnosis.

Androgen deprivation therapy (ADT) forms the cornerstone of treatment in locally advanced and metastatic prostate cancer (PCa). Since the growth hormone–insulin-like growth factor (GH–IGF-1) axis has been implicated in prostate tumorigenesis, we aimed to evaluate the association between IGF-1 and its binding proteins on outcomes in men with metastatic PCa treated with ADT, with or without docetaxel (D). We analyzed serum samples for IGF-1 and its family proteins from baseline, 6 months post-randomization, and at the time of progression in men enrolled to receive ADT +/− D in the phase 3 CHAARTED trial. The key outcomes were time to the development of castrate-resistant prostate cancer and overall survival (OS). About 560 patients had samples available for analysis. At 6 months, significant increases in IGF-BP1 (mean Δ+27.4%, P = 0.033), IGF-BP3 (mean Δ+10.3%, P < 0.001), and IGF-BP4 (mean Δ+31.1%, P < 0.001) were seen in the ADT + D group, while the ADT group showed an increase in IGF-BP3 (mean Δ+5.5%, P = 0.015). A higher IGF-1:IGF-BP1 ratio at baseline and after 6 months was associated with improved OS in both the ADT (baseline: hazard ratio (HR) = 0.77, P = 0.026; 6 months: HR = 0.83, P = 0.036) and ADT + D groups (baseline: HR = 0.78, P = 0.04; 6 months: HR = 0.81, P = 0.018). Patients with a log₁₀IGF-1:IGF-BP1 ratio >1.3 at baseline had improved OS when meta-analyzed with data from a prior cohort (HR = 0.71). A higher baseline and 6-month IGF-1:IGF-BP1 ratio was associated with better OS. Further exploration of the IGF-1 axis will be important to assess its role as a predictive biomarker and to target this axis in therapeutic trials.

Despite the successful combination of therapies improving survival of estrogen receptor α (ER+) breast cancer patients with metastatic disease, mechanisms for acquired endocrine resistance remain to be fully elucidated. The RNA binding protein HNRNPA2B1 (A2B1), a reader of N(6)-methyladenosine (m6A) in transcribed RNA, is upregulated in endocrine-resistant, ER+ LCC9 and LY2 cells compared to parental MCF-7 endocrine-sensitive luminal A breast cancer cells. The miRNA-seq transcriptome of MCF-7 cells overexpressing A2B1 identified the serine metabolic processes pathway. Increased expression of two key enzymes in the serine synthesis pathway (SSP), phosphoserine aminotransferase 1 (PSAT1) and phosphoglycerate dehydrogenase (PHGDH), correlates with poor outcomes in ER+ breast patients who received tamoxifen (TAM). We reported that PSAT1 and PHGDH were higher in LCC9 and LY2 cells compared to MCF-7 cells and their knockdown enhanced TAM sensitivity in these-resistant cells. Here we demonstrate that stable, modest overexpression of A2B1 in MCF-7 cells increased PSAT1 and PHGDH and endocrine resistance. We identified four miRNAs downregulated in MCF-7-A2B1 cells that directly target the PSAT1 3′UTR (miR-145-5p and miR-424-5p), and the PHGDH 3′UTR (miR-34b-5p and miR-876-5p) in dual luciferase assays. Lower expression of miR-145-5p and miR-424-5p in LCC9 and ZR-75-1-4-OHT cells correlated with increased PSAT1 and lower expression of miR-34b-5p and miR-876-5p in LCC9 and ZR-75-1-4-OHT cells correlated with increased PHGDH. Transient transfection of these miRNAs restored endocrine-therapy sensitivity in LCC9 and ZR-75-1-4-OHT cells. Overall, our data suggest a role for decreased A2B1-regulated miRNAs in endocrine resistance and upregulation of the SSP to promote tumor progression in ER+ breast cancer.

We assessed the efficacy and safety of combining bevacizumab with temsirolimus in patients with advanced extra-pancreatic neuroendocrine tumors. This NCI-sponsored multicenter, open-label, phase II study (NCT01010126) enrolled patients with advanced, recurrent, or metastatic extra-pancreatic neuroendocrine tumors. All patients were treated with temsirolimus and bevacizumab until disease progression or unacceptable toxicity. Temsirolimus 25 mg was administered i.v. on days 1, 8, 15, and 22 and bevacizumab 10 mg/kg i.v. on days 1 and 15 of a 4-week cycle. Discontinuation of temsirolimus or bevacizumab did not require discontinuation of the other agent. The primary endpoints were objective response rate and 6-month progression-free survival rate. Fifty-nine patients were enrolled in this study, and 54 were evaluated for efficacy and adverse events. While median progression-free survival was 7.1 months, the median duration of treatment with temsirolimus was 3.9 months and that with bevacizumab was 3.5 months. The objective response rate of combination therapy was 2%, and 6-month progression-free survival was 48%. The most frequently reported grade 3–4 adverse events included fatigue (13%), hypertension (13%), and bleeding (13%). Close to 54% of the patients discontinued treatment due to adverse events, refusal of further treatment, or treatment delays. Three deaths occurred in the study, of which two were due to treatment-related bowel perforations. Given the minimal efficacy and increased toxicity seen with the combination of bevacizumab and temsirolimus, we do not recommend the use of this regimen in patients with advanced extra-pancreatic neuroendocrine tumors.

Molecular testing contributes to improving the diagnosis of indeterminate thyroid nodules (ITNs). ThyroidPrint® is a ten-gene classifier aimed to rule out malignancy in ITN. Post-validation studies are necessary to determine the real-world clinical benefit of ThyroidPrint® in patients with ITN. A single-center, prospective, noninterventional clinical utility study was performed, analyzing the impact of ThyroidPrint® in the physicians’ clinical decisions for ITN. Demographics, nodule characteristics, benign call rates (BCRs), and surgical outcomes were measured. Histopathological data were collected from surgical biopsies of resected nodules. Of 1272 fine-needle aspirations, 109 (8.6%) were Bethesda III and 135 (10.6%) were Bethesda IV. Molecular testing was performed in 155 of 244 ITN (63.5%), of which 104 were classified as benign (BCR of 67.1%). After a median follow-up of 15 months, 103 of 104 (99.0%) patients with a benign ThyroidPrint® remained under surveillance and one patient underwent surgery which was a follicular adenoma. Surgery was performed in all 51 patients with a suspicious for malignancy as per ThyroidPrint® result and in 56 patients who did not undergo testing, with a rate of malignancy of 70.6% and 32.1%, respectively. A higher BCR was observed in follicular lesion of undetermined significance (87%) compared to atypia of undetermined significance (58%) (P < 0.05). False-positive cases included four benign follicular nodules and six follicular and four oncocytic adenomas. Our results show that, physicians chose active surveillance instead of diagnostic surgery in all patients with a benign ThyroidPrint® result, reducing the need for diagnostic surgery in 67% of patients with preoperative diagnosis of ITN.