RESUMEN
Precise genome engineering of living cells has been revolutionized by the introduction of the highly specific and easily programmable properties of the clustered regularly interspaced short palindromic repeats (CRISPR) 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 knock-in 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 knock-in 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 mutant FOXA1 protein expression and intended amino acid substitutions via western blotting and mass spectrometry. In addition, we show that mutant allele frequency estimation is easily achieved by topoisomerase-based 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 wild-type FOXA1 with precise knock-in mutants to enable the systematic analysis of its molecular mechanisms within the appropriate physiological context.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Factor Nuclear 3-alfa del Hepatocito/genética , Neoplasias/genética , Alelos , Humanos , Mutagénesis , Mutación , Neoplasias/patologíaRESUMEN
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 PDGFRß) 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.