Outcomes for men with localized prostate cancer vary widely, with some men effectively managed without treatment on active surveillance, while other men rapidly progress to metastatic disease despite curative-intent therapies. One of the strongest prognostic indicators of outcome is grade groups based on the Gleason grading system. Gleason grade 4 prostate cancer with cribriform morphology is associated with adverse outcomes and can be utilized clinically to improve risk stratification. The underpinnings of disease aggressiveness associated with cribriform architecture are not fully understood. Most studies have focused on genetic and molecular alterations in cribriform tumor cells; however, less is known about the tumor microenvironment in cribriform prostate cancer. Cancer-associated fibroblasts (CAFs) are a heterogeneous population of fibroblasts in the tumor microenvironment that impact cancer aggressiveness. The overall goal of this study was to determine if cribriform prostate cancers are associated with a unique repertoire of CAFs. Radical prostatectomy whole-tissue sections were analyzed for the expression of fibroblast markers (ASPN in combination with FAP, THY1, ENG, NT5E, TNC, and PDGFRβ) in stroma adjacent to benign glands and in Gleason grade 3, Gleason grade 4 cribriform, and Gleason grade 4 noncribriform prostate cancer by RNAscope ® . Halo ® Software was used to quantify percent positive stromal cells and expression per positive cell. The fibroblast subtypes enriched in prostate cancer were highly heterogeneous. Both overlapping and distinct populations of low abundant fibroblast subtypes in benign prostate stroma were enriched in Gleason grade 4 prostate cancer with cribriform morphology compared to Gleason grade 4 prostate cancer with noncribriform morphology and Gleason grade 3 prostate cancer. In addition, gene expression was distinctly altered in CAF subtypes adjacent to cribriform prostate cancer. Overall, these studies suggest that cribriform prostate cancer has a unique tumor microenvironment that may distinguish it from other Gleason grade 4 morphologies and lower Gleason grades.
Precise genome engineering of living cells has been revolutionized by the introduction of the highly specific and easily programmable properties of CRISPR-Cas9 technology. This has greatly accelerated research into human health and has facilitated the discovery of novel therapeutics. CRISPR-Cas9 is most widely employed for its ability to inactivate or knockout specific genes, but can be also used to introduce subtle site-specific substitutions of DNA sequences that can lead to changes in the amino acid composition of proteins. Despite the proven success of CRISPR-based knockin strategies of genes in typical diploid cells (i.e. cells containing two sets of chromosomes), precise editing of cancer cells, that typically have unstable genomes and multiple copies of chromosomes, is more challenging and not adequately addressed in the literature. Herein, we detail our methodology for replacing endogenous proteins with intended knockin mutants in polyploid cancer cells and discuss our experimental design, screening strategy, and facile allele-frequency estimation methodology. As proof of principle, we performed genome editing of specific amino acids within the pioneer transcription factor FOXA1, a critical component of estrogen and androgen receptor signaling,
Precise genome engineering of living cells has been revolutionized by the introduction of the highly specific and easily programmable properties of CRISPR-Cas9 technology. This has greatly accelerated research into human health and has facilitated the discovery of novel therapeutics. CRISPR-Cas9 is most widely employed for its ability to inactivate, or knockout, specific genes, but can be also used to introduce subtle site-specific substitutions of DNA sequences that can lead to changes in the amino acid composition of proteins. Despite the proven success of CRISPR-based knockin strategies of genes in typical diploid cells (i.e. cells containing two sets of chromosomes), precise editing of cancer cells, that typically have unstable genomes and multiple copies of chromosomes, is more challenging and not adequately addressed in the literature. Herein we detail our methodology for replacing endogenous proteins with intended knockin mutants in polyploid cancer cells and discuss our experimental design, screening strategy, and facile allele-frequency estimation methodology. As proof of principle, we performed genome editing of specific amino acids within the pioneer transcription factor FOXA1, a critical component of estrogen and androgen receptor signaling, in MCF-7 breast cancer cells. We confirm proper levels of mutant FOXA1 protein expression and intended amino acids substitutions via western blotting and mass spectrometry. In addition, we show that mutant allele-frequency estimation is easily achieved by TOPO cloning combined with allele-specific PCR, which we later confirmed by next-generation RNA-sequencing. Typically, there are 4 - 5 copies (alleles) of FOXA1 in breast cancer cells making the editing of this protein inherently challenging. As a result, most studies that focus on FOXA1 mutants rely on ectopic overexpression of FOXA1 from a plasmid. Therefore, we provide an optimized methodology for replacing endogenous wildtype FOXA1 with precise knockin mutants to enable the systematic analysis of its molecular mechanisms within the appropriate physiological context.
ER-positive breast cancers compose most breast cancers at the time of diagnosis and are primarily driven by mitogenic estrogen signaling. In ER-positive breast cancers, the pioneer transcription factor FOXA1 plays a critical role in the estrogen receptor (ER) function. It binds to condensed chromatin and promotes chromatin accessibility for subsequent ER binding upon estrogen stimulation. We have reported that TNFa-stimulated proinflammatory signaling relocates FOXA1 to a new set of latent enhancers, which initiates the binding of estrogen liganded ER and subsequent expression of a unique transcriptome with clinical significance. The redistribution of FOXA1 occurs within 40 mins of the TNFa treatment, which implies a rapid signaling cascade that arises from changes to either FOXA1’s post-translational modifications (PTMs) or its binding partners. To understand this genomic redistribution of FOXA1, we compared the post-translational modifications (PTMs) of FOXA1 from Vehicle, E2, TNFa, and E2+TNFa treated MCF-7 breast cancer cells. More than five acetylation and phosphorylation events have been identified around the DNA binding domain of FOXA1 by semi-quantitative and quantitative mass spectrometry approaches, and their abundance varies across treatments. To study these PTMs of FOXA1, we used CRISPR/Cas9 to create specific knock-in mutations to mimic or prevent acetylation events in MCF-7 cells. Specifically, we engineered MCF-7 cell lines where K270 was mutated to glutamine (K270Q) to mimic acetylation. And for comparison, we also created cell lines where K270 was mutated to arginine (K270R) to prevent acetylation of FOXA1. Our data, including FOXA1 ChIP-seq and RNA-seq, revealed the genomic redistribution of FOXA1 with these PTMs, which subsequently alters gene expression programs and promotes cell growth, migration, or chemoresistance. These results were confirmed in other ER+ cell lines (such as T47D cells) providing evidence for the generalizability of our findings. Taken together, our data suggest that inflammatory signaling signaling can reshape the enhancer landscape of FOXA1 through post-translational modifications, resulting in changes to estrogen signaling that have profound effects on breast cancer biology. Citation Format: Shen Li, Hector L. Franco, Hyunsoo Kim, Rosemary N. Plagens, Raul Mendez-Giraldez, Colby Tubbs, Venkat Malladi, Joseph Garay. Proinflammatory and estrogen signaling modulates the chemoresistance and metastasis of breast cancer cells through post-translational modifications of pioneering factor FOXA1 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-03.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.