WCRC-25: A novel luminal Invasive Lobular Carcinoma cell line model.

Breast cancer is categorized by the molecular and histologic presentation of the tumor, with the major histologic subtypes being No Special Type (NST) and Invasive Lobular Carcinoma (ILC). ILC are characterized by growth in a single file discohesive manner with stromal infiltration attributed to their hallmark pathognomonic loss of E-cadherin ( CDH1 ). Few ILC cell line models are available to researchers. Here we report the successful establishment and characterization of a novel ILC cell line, WCRC-25, from a metastatic pleural effusion from a postmenopausal Caucasian woman with metastatic ILC. WCRC-25 is an ER-negative luminal epithelial ILC cell line with both luminal and Her2-like features. It exhibits anchorage independent growth and haptotactic migration towards Collagen I. Sequencing revealed a CDH1 Q706* truncating mutation, together with mutations in FOXA1, CTCF, BRCA2 and TP53 , which were also seen in a series of metastatic lesions from the patient. Copy number analyses revealed amplification and deletion of genes frequently altered in ILC while optical genome mapping revealed novel structural rearrangements. RNA-seq analysis comparing the primary tumor, metastases and the cell line revealed signatures for cell cycle progression and receptor tyrosine kinase signaling. To assess targetability, we treated WCRC-25 with AZD5363 and Alpelisib confirming WCRC-25 as susceptible to PI3K/AKT signaling inhibition as predicted by our RNA sequencing analysis. In conclusion, we report WCRC-25 as a novel ILC cell line with promise as a valuable research tool to advance our understanding of ILC and its therapeutic vulnerabilities. Financial support: The work was in part supported by a Susan G Komen Leadership Grant to SO (SAC160073) and NCI R01 CA252378 (SO/AVL). AVL and SO are Komen Scholars, Hillman Foundation Fellows and supported by BCRF. This project used the UPMC Hillman Cancer Center and Tissue and Research Pathology/Pitt Biospecimen Core shared resource which is supported in part by award P30CA047904. This research was also supported in part by the University of Pittsburgh Center for Research Computing, RRID:SCR_022735, through the resources provided. Specifically, this work used the HTC cluster, which is supported by NIH award number S10OD028483. Finally, partial support was provided by the Magee-Womens Research Institute and Foundation, The Shear Family Foundation, and The Metastatic Breast Cancer Network.

In cis TP53 and RAD51C pathogenic variants may predispose to sebaceous gland carcinomas.

Pathogenic variants in TP53 have been classically thought to cause Li-Fraumeni syndrome (LFS), a cancer predisposition with high risks for various childhood- and adult-onset malignancies. However, increased genetic testing has lately revealed, that pathogenic variant carriers exhibit a broader range of phenotypes and that penetrance may be dependent both on variant type and modifiers. Using next generation sequencing and short tandem repeat analysis, we identified germline pathogenic variants in TP53 and RAD51C located in cis on chromosome 17 in a 43-year-old male, who has developed a rare sebaceous gland carcinoma (SGC) but so far no tumors of the LFS spectrum. This course mirrors a Trp53-Rad51c-double-mutant cis mouse-model, which similarly develops SGC, while the characteristic Trp53-associated tumor spectrum occurs with significantly lower frequency. Therefore, we propose that co-occurent pathogenic variants in RAD51C and TP53 may predispose to SGC, reminiscent of Muir-Torre syndrome. Further, this report supports the diversity of clinical presentations associated with germline TP53 alterations, and thus, the proposed expansion of LFS to heritable TP53-related cancer syndrome.

Differential Regulation and Targeting of Estrogen Receptor α Turnover in Invasive Lobular Breast Carcinoma.

Invasive lobular breast carcinoma (ILC) accounts for 10% to 15% of breast cancers diagnosed annually. Evidence suggests that some aspects of endocrine treatment response might differ between invasive ductal carcinoma (IDC) and ILC, and that patients with ILC have worse long-term survival. We analyzed The Cancer Genome Atlas dataset and observed lower levels of ESR1 mRNA (P = 0.002) and ERα protein (P = 0.038) in ER+ ILC (n = 137) compared to IDC (n = 554), and further confirmed the mRNA difference in a local UPMC cohort (ILC, n = 143; IDC, n = 877; P < 0.005). In both datasets, the correlation between ESR1 mRNA and ERα protein was weaker in ILC, suggesting differential post-transcriptional regulation of ERα. In vitro, 17ß-estradiol (E2) decreased the rate of degradation and increased the half-life of ERα in ILC cell lines, whereas the opposite was observed in IDC cell lines. Further, E2 failed to induce robust ubiquitination of ERα in ILC cells. To determine the potential clinical relevance of these findings, we evaluated the effect of 2 selective estrogen receptor downregulators (SERDs), ICI 182,780 and AZD9496, on ERα turnover and cell growth. While ICI 182,780 and AZD9496 showed similar effects in IDC cells, in ILC cell lines, AZD9496 was not as effective as ICI 182,780 in decreasing ERα stability and E2-induced proliferation. Furthermore, AZD9496 exhibited partial agonist activity in growth assays in ILC cell lines. Our study provides evidence for a distinct ERα regulation by SERDs in ILC cell lines, and therefore it is important to include ILC models into preclinical and clinical testing of novel SERDs.

Spatial and developmental synthesis of endogenous sesquiterpene lactones supports function in growth regulation of sunflower.

MAIN CONCLUSION: Tissue-specific occurrence and formation of endogenous sesquiterpene lactones has been assessed and suggests physiological function as antagonists of auxin-induced plant growth in sunflower. Sunflower, Helianthus annuus, accumulate high concentrations of bioactive sesquiterpene lactones (STL) in glandular trichomes, but in addition, structurally different STL occur in only trace amounts in the inner tissues. The spatial and temporal production of these endogenous STL during early phases of plant development is widely unknown and their physiological function as putative natural growth regulators is yet speculative. By means of HPLC and MS analysis it was shown that costunolide, dehydrocostuslactone, 8-epixanthatin and tomentosin are already present in dry seeds and can be extracted in low amounts from cotyledons, hypocotyls and roots of seedlings during the first days after germination. Semi-quantitative and RT-qPCR experiments with genes of the key enzymes of two independent routes of the endogenous STL biosynthesis confirmed the early and individual expression in these organs and revealed a gradual down regulation during the first 72-96 h after germination. Light irradiation of the plants led to a fast, but transient increase of STL in parts of the hypocotyl which correlated with growth retardation of the stem. One-sided external application of costunolide on hypocotyls conferred reduced growth of the treated side, thus resulting in the curving of the stem towards the side of the application. This indicates the inhibiting effects of STL on plant growth. The putative function of endogenous STL in sunflower as antagonists of auxin in growth processes is discussed.