Lymph/angiogenesis contribute to sex differences in lung cancer through ERalpha signalling.

Endocr Relat Cancer.

2018 Nov 01

Dubois C, Rocks N, Blacher S, Primac I, Gallez A, García-Caballero M, Gérard C, Brouchet L, Noel A, Lenfant F, Cataldo D, Péqueux C.
PMID: 30444717 | DOI: 10.1530/ERC-18-0328

Estrogen signalling pathways are emerging targets for lung cancer therapy. Unravelling the contribution of estrogens in lung cancer development is a pre-requisite to support the development of sex-based treatments and to identify patients who could potentially benefit from anti-estrogen treatments. In this study, we highlight the contribution of lymphatic and blood endothelia in the sex-dependent modulation of lung cancer. The orthotopic graft of syngeneic lung cancer cells into immunocompetent mice showed that lung tumours grew faster in female mice than in males. Moreover, estradiol (E2) promoted tumour development in female mice and increased lymph/angiogenesis and levels of VEGFA and bFGF in lung tumours of females through an estrogen receptor (ER) alpha-dependent pathway. Furthermore, while treatment with ERbeta antagonist was inefficient, ERalpha antagonist (MPP) and tamoxifen decreased lung tumour volumes, altered blood and lymphatic vasculature and reduced VEGFA and bFGF levels in females, but not in males. Finally, the quantification of lymphatic and blood vasculature of lung adenocarcinoma biopsies from patients aged between 35 to 55 years old revealed more extensive lymphangiogenesis and angiogenesis in tumour samples issued from women than from men. In conclusion, our findings highlight an E2/ERalpha-dependent modulation of lymphatic and blood vascular components of lung tumour microenvironment. Our study has potential clinical implication in a personalised medicine perspective by pointing to the importance of estrogen status or supplementation on lung cancer development that should be considered to adapt therapeutic strategies.

Viral and Genomic Drivers of Squamous Cell Neoplasms Arising in the Lacrimal Drainage System

Cancers

2022 May 23

Ramberg, I;Vieira, FG;Toft, PB;von Buchwald, C;Heegaard, S;
PMID: 35626161 | DOI: 10.3390/cancers14102558

The pathogenesis of squamous cell neoplasms arising in the lacrimal drainage system is poorly understood, and the underlying genomic drivers for disease development remain unexplored. We aimed to investigate the genomic aberrations in carcinomas arising in the LDS and correlate the findings to human papillomavirus (HPV) status. The HPV analysis was performed using HPV DNA PCR, HPV E6/E7 mRNA in-situ hybridization, and p16 immunohistochemistry. The genomic characterization was performed by targeted DNA sequencing of 523 cancer-relevant genes. Patients with LDS papilloma (n = 17) and LDS carcinoma (n = 15) were included. There was a male predominance (68%) and a median age at diagnosis of 46.0 years (range 27.5-65.5 years) in patients with papilloma and 63.8 years (range 34.0-87.2 years) in patients with carcinoma. Transcriptional activity of the HPV E6/E7 oncogenes was detected in the whole tumor thickness in 12/15 (80%) papillomas (HPV6, 11, 16) and 10/15 (67%) squamous cell carcinomas (SCC) (HPV11: 3/15 (20%) and HPV16: 7/15 (47%)). Pathogenic variants in PIK3CA, FGFR3, AKT1, and PIK3R1, wildtype TP53, p16 overexpression, and deregulated high-risk E6/E7 transcription characterized the HPV16-positive SCC. The deregulated pattern of HPV E6/E7 expression, correlating with HPV DNA presence and p16 positivity, supports a causal role of HPV in a subset of LDS papillomas and carcinomas. The viral and molecular profile of LDS SCC resembles that of other HPV-driven SCC.

Quantitative in situ measurement of estrogen receptor mRNA predicts response to tamoxifen.

PLoS One, 7(5):e36559.

Bordeaux JM, Cheng H, Welsh AW, Haffty BG, Lannin DR, Wu X, Su N, Ma XJ, Luo Y, Rimm DL. (2012).
PMID: 22606272 | DOI: 10.1371/journal.pone.0036559.

PURPOSE: Quantification of mRNA has historically been done by reverse transcription polymerase chain reaction (RT-PCR). Recently, a robust method of detection of mRNA utilizing in situ hybridization has been described that is linear and shows high specificity with low background. Here we describe the use of the AQUA method of quantitative immunofluorescence (QIF) for measuring mRNA in situ using ESR1 (the estrogen receptor alpha gene) in breast cancer to determine its predictive value compared to Estrogen Receptor α (ER) protein. METHODS: Messenger RNA for ER (ESR1) and Ubiquitin C (UbC) were visualized using RNAscope probes and levels were quantified by quantitative in situ hybridization (qISH) on two Yale breast cancer cohorts on tissue microarrays. ESR1 levels were compared to ER protein levels measured by QIF using the SP1 antibody. RESULTS: ESR1 mRNA is reproducibly and specifically measurable by qISH on tissue collected from 1993 or later. ESR1 levels were correlated to ER protein levels in a non-linear manner on two Yale cohorts. High levels of ESR1 were found to be predictive of response to tamoxifin. CONCLUSION: Quantification of mRNA using qISH may allow assessment of large cohorts with minimal formalin fixed, paraffin embedded tissue. Exploratory data using this method suggests that measurement of ESR1 mRNA levels may be predictive of response to endocrine therapy in a manner that is different from the predictive value of ER.

Pin1 modulates ERα levels in breast cancer through inhibition of phosphorylation-dependent ubiquitination and degradation.

Oncogene. 2014 Mar 13;33(11):1438-47.

Rajbhandari P, Schalper KA, Solodin NM, Ellison-Zelski SJ, Ping Lu K, Rimm DL, Alarid ET (2013).
PMID: 23542176 | DOI: 10.1038/onc.2013.78.

Estrogen receptor-alpha (ERα) is an important biomarker used to classify and direct therapy decisions in breast cancer (BC). Both ERα protein and its transcript, ESR1, are used to predict response to tamoxifen therapy, yet certain tumors have discordant levels of ERα protein and ESR1, which is currently unexplained. Cellular ERα protein levels can be controlled post-translationally by the ubiquitin-proteasome pathway through a mechanism that depends on phosphorylation at residue S118. Phospho-S118 (pS118-ERα) is a substrate for the peptidyl prolyl isomerase, Pin1, which mediates cis-trans isomerization of the pS118-P119 bond to enhance ERα transcriptional function. Here, we demonstrate that Pin1 can increase ERα protein without affecting ESR1 transcript levels by inhibiting proteasome-dependent receptor degradation. Pin1 disrupts ERα ubiquitination by interfering with receptor interactions with the E3 ligase, E6AP, which also is shown to bind pS118-ERα. Quantitative in situ assessments of ERα protein, ESR1, and Pin1 in human tumors from a retrospective cohort show that Pin1 levels correlate with ERα protein but not to ESR1 levels. These data show that ERα protein is post-translationally regulated by Pin1 in a proportion of breast carcinomas. As Pin1 impacts both ERα protein levels and transactivation function, these data implicate Pin1 as a potential surrogate marker for predicting outcome of ERα-positive BC.

Estrogen Receptor-α Quantification in Breast Cancer: Concordance Between Immunohistochemical Assays and mRNA-In Situ Hybridization for ESR1 Gene.

Appl Immunohistochem Mol Morphol.

2019 Mar 27

Thomsen C, Nielsen S, Nielsen BS, Pedersen SH, Vyberg M.
PMID: 30920963 | DOI: 10.1097/PAI.0000000000000760

Immunohistochemical (IHC) quantification of estrogen receptor-α (ER) is used for assessment of treatment regimen in breast cancer. Different ER IHC assays may produce diverging results, because of different antibody clones, protocols, and stainer platforms. Objective tissue-based techniques to assess sensitivity and specificity of IHC assays are therefore needed. We tested the usability of ER mRNA-in situ hybridization (mRNA-ISH) in comparison with assays based on clones SP1 and 6F11. We selected 56 archival specimens according to their reported ER IHC positivity, representing a wide spectrum from negative to strongly positive cases. The specimens were used to prepare 4 TMAs with 112 cores. Serial sections of each TMA were stained for ER and pan-cytokeratin (PCK) by IHC and ESR1 (ER gene) by mRNA-ISH. Digital image analysis (DIA) was used to determine ER IHC H-score. ESR1 mRNA-ISH was scored both manually and by DIA. DIA showed a nonlinear correlation between IHC and ESR1 mRNA-ISH with R-values of 0.80 and 0.78 for the ER antibody clones SP1 and 6F11, respectively. Comparison of manual mRNA-ISH scoring categories and SP1 and 6F11 IHC H-scores showed a highly significant relationship (P<0.001). In conclusion, the study showed good correlation between mRNA-ISH and IHC, suggesting that mRNA-ISH can be a valuable tool in the assessment of the sensitivity and specificity of ER IHC assays.

Single-Cell Transcriptome Analysis Reveals Estrogen Signaling Coordinately Augments One-Carbon, Polyamine, and Purine Synthesis in Breast Cancer.

Cell Rep.

2018 Nov 20

Zhu D, Zhao Z, Cui G, Chang S, Hu L, See YX, Lim MGL, Guo D, Chen X, Robson P, Luo Y, Cheung E.
PMID: 30463022 | DOI: 10.1016/j.celrep.2018.10.093

Estrogen drives breast cancer (BCa) progression by directly activating estrogen receptor α (ERα). However, because of the stochastic nature of gene transcription, it is important to study the estrogen signaling pathway at the single-cell level to fully understand how ERα regulates transcription. Here, we performed single-cell transcriptome analysis on ERα-positive BCa cells following 17β-estradiol stimulation and reconstructed the dynamic estrogen-responsive transcriptional network from discrete time points into a pseudotemporal continuum. Notably, differentially expressed genes show an estrogen-stimulated metabolic switch that favors biosynthesis but reduces estrogen degradation. Moreover, folate-mediated one-carbon metabolism is reprogrammed through the mitochondrial folate pathway and polyamine and purine synthesis are upregulated coordinately. Finally, we show AZIN1 and PPAT are direct ERα targets that are essential for BCa cell survival and growth. In summary, our study highlights the dynamic transcriptional heterogeneity in ERα-positive BCa cells upon estrogen stimulation and uncovers a mechanism of estrogen-mediated metabolic switch.

Molecular Analysis of HPV-independent Primary Endometrial Squamous Cell Carcinoma Reveals TP53 and CDKN2A comutations: A Clinicopathologic Analysis With Re-evaluation of Diagnostic Criteria

The American journal of surgical pathology

2022 Sep 05

Hopkins, MR;Palsgrove, DN;Ronnett, BM;Vang, R;Lin, J;Murdock, TA;
PMID: 36069815 | DOI: 10.1097/PAS.0000000000001970

Human papillomavirus (HPV)-independent primary endometrial squamous cell carcinoma (PESCC) is a rare but aggressive subtype of endometrial carcinoma for which little is known about the genomic characteristics. Traditional criteria have restricted the diagnosis of PESCC to cases without any cervical involvement. However, given that modern ancillary techniques can detect HPV and characteristic genetic alterations that should identify the more common mimics in the differential diagnosis, including endometrial endometrioid carcinoma with extensive squamous differentiation and HPV-associated primary cervical squamous cell carcinoma, those criteria may benefit from revision. To further characterize PESCC, we identified 5 cases of pure squamous cell carcinoma dominantly involving the endometrium that had the potential to be PESCC: 1 case involving only the endometrium and 4 cases with some involvement of the cervix. Clinicopathologic features were assessed and immunohistochemical analysis (p16, estrogen receptor, progesterone receptor, and p53), HPV RNA in situ hybridization (high-risk and low-risk cocktails and targeted probes for 16 and 18), and molecular studies were performed. All tumors showed aberrant/mutation-type p53 expression, were negative for estrogen receptor, progesterone receptor, and p16, and had no detectable HPV. Per whole-exome sequencing, 4 of the 5 tumors demonstrated comutations in TP53 and CDKN2A (p16). Four patients died of disease within 20 months (range, 1 to 20 mo; mean, 9 mo), and 1 patient had no evidence of disease at 38 months. PESCC represents a unique, clinically aggressive subtype of endometrial cancer with TP53 and CDKN2A comutations. This characteristic profile, which is similar to HPV-independent squamous cell carcinoma of the vulva, is distinct from endometrioid carcinoma with extensive squamous differentiation and HPV-associated primary cervical squamous cell carcinoma and can be used to distinguish PESCC from those mimics even when cervical involvement is present. Diagnostic criteria for PESCC should be relaxed to allow for cervical involvement when other pathologic features are consistent with, and ancillary techniques are supportive of classification as such.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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