Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue.

We describe the use of a ligation-based targeted whole transcriptome expression profiling assay, TempO-Seq, to profile formalin-fixed paraffin-embedded (FFPE) tissue, including H&E stained FFPE tissue, by directly lysing tissue scraped from slides without extracting RNA or converting the RNA to cDNA. The correlation of measured gene expression changes in unfixed and fixed samples using blocks prepared from a pellet of a single cell type was R2 = 0.97, demonstrating that no significant artifacts were introduced by fixation. Fixed and fresh samples prepared in an equivalent manner produced comparable sequencing depth results (+/- 20%), with similar %CV (11.5 and 12.7%, respectively), indicating no significant loss of measurable RNA due to fixation. The sensitivity of the TempO-Seq assay was the same whether the tissue section was fixed or not. The assay performance was equivalent for human, mouse, or rat whole transcriptome. The results from 10 mm2 and 2 mm2 areas of tissue obtained from 5 µm thick sections were equivalent, thus demonstrating high sensitivity and ability to profile focal areas of histology within a section. Replicate reproducibility of separate areas of tissue ranged from R2 = 0.83 (lung) to 0.96 (liver) depending on the tissue type, with an average correlation of R2 = 0.90 across nine tissue types. The average %CVs were 16.8% for genes expressed at greater than 200 counts, and 20.3% for genes greater than 50 counts. Tissue specific differences in gene expression were identified and agreed with the literature. There was negligible impact on assay performance using FFPE tissues that had been archived for up to 30 years. Similarly, there was negligible impact of H&E staining, facilitating accurate visualization for scraping and assay of small focal areas of specific histology within a section.

Epigenetic and Transcriptomic Profiling of Mammary Gland Development and Tumor Models Disclose Regulators of Cell State Plasticity.

Cell state reprogramming during tumor progression complicates accurate diagnosis, compromises therapeutic effectiveness, and fuels metastatic dissemination. We used chromatin accessibility assays and transcriptional profiling during mammary development as an agnostic approach to identify factors that mediate cancer cell state interconversions. We show that fetal and adult basal cells share epigenetic features consistent with multi-lineage differentiation potential. We find that DNA-binding motifs for SOX transcription factors are enriched in chromatin that is accessible in stem/progenitor cells and inaccessible in differentiated cells. In both mouse and human tumors, SOX10 expression correlates with stem/progenitor identity, dedifferentiation, and invasive characteristics. Strikingly, we demonstrate that SOX10 binds to genes that regulate neural crest cell identity, and that SOX10-positive tumor cells exhibit neural crest cell features.

Single-Cell Transcriptomes Distinguish Stem Cell State Changes and Lineage Specification Programs in Early Mammary Gland Development.

The mammary gland consists of cells with gene expression patterns reflecting their cellular origins, function, and spatiotemporal context. However, knowledge of developmental kinetics and mechanisms of lineage specification is lacking. We address this significant knowledge gap by generating a single-cell transcriptome atlas encompassing embryonic, postnatal, and adult mouse mammary development. From these data, we map the chronology of transcriptionally and epigenetically distinct cell states and distinguish fetal mammary stem cells (fMaSCs) from their precursors and progeny. fMaSCs show balanced co-expression of factors associated with discrete adult lineages and a metabolic gene signature that subsides during maturation but reemerges in some human breast cancers and metastases. These data provide a useful resource for illuminating mammary cell heterogeneity, the kinetics of differentiation, and developmental correlates of tumorigenesis.

Lgr5 is a marker for fetal mammary stem cells, but is not essential for stem cell activity or tumorigenesis.

The search for the bipotent mammary stem cells that drive mammary development requires markers to enable their prospective isolation. There is general agreement that bipotent mouse mammary stem cells are abundant in late fetal development, but their existence in the adult is vigorously debated. Among markers useful for mammary stem cell identification, the Wnt co-receptor Lgr5 has been suggested by some to be both "necessary and sufficient" for bipotency and transplantation of adult mammary stem cell activity, though other studies disagree. Importantly, the relevance of Lgr5 to the bipotency of fetal mammary stem cells has not been studied. We show here that expression of a fluorescent protein driven by the endogenous Lgr5 promoter enables significant fetal mammary stem cell enrichment. We used lineage tracing to demonstrate embryonic cells expressing Lgr5 are bipotent, while their adult counterparts are myoepithelial restricted. Importantly, our data conclusively demonstrate that Lgr5 is dispensable for both fetal and adult mammary stem cell activity and for the development of mammary tumors.

PIK3CA(H1047R) Accelerates and Enhances KRAS(G12D)-Driven Lung Tumorigenesis.

KRAS-activating mutations drive human non-small cell lung cancer and initiate lung tumorigenesis in genetically engineered mouse (GEM) models. However, in a GEM model of KRAS(G12D)-induced lung cancer, tumors arise stochastically following a latency period, suggesting that additional events are required to promote early-stage tumorigenic expansion of KRAS(G12D)-mutated cells. PI3Kα (PIK3CA) is a direct effector of KRAS, but additional activation of PI3'-lipid signaling may be required to potentiate KRAS-driven lung tumorigenesis. Using GEM models, we tested whether PI3'-lipid signaling was limiting for the promotion of KRAS(G12D)-driven lung tumors by inducing the expression of KRAS(G12D) in the absence and presence of the activating PIK3CA(H1047R) mutation. PIK3CA(H1047R) expression alone failed to promote tumor formation, but dramatically enhanced tumorigenesis initiated by KRAS(G12D). We further observed that oncogenic cooperation between KRAS(G12D) and PIK3CA(H1047R) was accompanied by PI3Kα-mediated regulation of c-MYC, GSK3ß, p27(KIP1), survivin, and components of the RB pathway, resulting in accelerated cell division of human or mouse lung cancer-derived cell lines. These data suggest that, although KRAS(G12D) may activate PI3Kα by direct biochemical mechanisms, PI3'-lipid signaling remains rate-limiting for the cell-cycle progression and expansion of early-stage KRAS(G12D)-initiated lung cells. Therefore, we provide a potential mechanistic rationale for the selection of KRAS and PIK3CA coactivating mutations in a number of human malignancies, with implications for the clinical deployment of PI3' kinase-targeted therapies.

Sox10 Regulates Stem/Progenitor and Mesenchymal Cell States in Mammary Epithelial Cells.

To discover mechanisms that mediate plasticity in mammary cells, we characterized signaling networks that are present in the mammary stem cells responsible for fetal and adult mammary development. These analyses identified a signaling axis between FGF signaling and the transcription factor Sox10. Here, we show that Sox10 is specifically expressed in mammary cells exhibiting the highest levels of stem/progenitor activity. This includes fetal and adult mammary cells in vivo and mammary organoids in vitro. Sox10 is functionally relevant, as its deletion reduces stem/progenitor competence whereas its overexpression increases stem/progenitor activity. Intriguingly, we also show that Sox10 overexpression causes mammary cells to undergo a mesenchymal transition. Consistent with these findings, Sox10 is preferentially expressed in stem- and mesenchymal-like breast cancers. These results demonstrate a signaling mechanism through which stem and mesenchymal states are acquired in mammary cells and suggest therapeutic avenues in breast cancers for which targeted therapies are currently unavailable.

Mutationally activated PIK3CA(H1047R) cooperates with BRAF(V600E) to promote lung cancer progression.

Adenocarcinoma of the lung, a leading cause of cancer death, frequently displays mutational activation of the KRAS proto-oncogene but, unlike lung cancers expressing mutated EGFR, ROS1, or ALK, there is no pathway-targeted therapy for patients with KRAS-mutated lung cancer. In preclinical models, expression of oncogenic KRAS(G12D) in the lung epithelium of adult mice initiates development of lung adenocarcinoma through activation of downstream signaling pathways. In contrast, mutationally activated BRAF(V600E), a KRAS effector, fails to initiate lung carcinogenesis despite highly efficient induction of benign lung tumorigenesis. To test if phosphoinositide 3-kinase (PI3K)-α (PIK3CA), another KRAS effector, might cooperate with oncogenic BRAF(V600E) to promote lung cancer progression, we used mice carrying a conditional allele of Pik3ca that allows conversion of the wild-type catalytic subunit of PIK3CA to mutationally activated PIK3CA(H1047R). Although expression of PIK3CA(H1047R) in the lung epithelium, either alone or in combination with PTEN silencing, was without phenotype, concomitant expression of BRAF(V600E) and PIK3CA(H1047R) led to dramatically decreased tumor latency and increased tumor burden compared with BRAF(V600E) alone. Most notably, coexpression of BRAF(V600E) and PIK3CA(H1047R) elicited lung adenocarcinomas in a manner reminiscent of the effects of KRAS(G12D). These data emphasize a role for PI3K signaling, not in lung tumor initiation per se, but in both the rate of tumor growth and the propensity of benign lung tumors to progress to a malignant phenotype. Finally, biologic and biochemical analysis of BRAF(V600E)/PIK3CA(H1047R)-expressing mouse lung cancer cells revealed mechanistic clues about cooperative regulation of the cell-division cycle and apoptosis by these oncogenes.

A central role for RAF→MEK→ERK signaling in the genesis of pancreatic ductal adenocarcinoma.

UNLABELLED: KRAS mutation is a hallmark of pancreatic ductal adenocarcinoma (PDA) but remains an intractable pharmacologic target. Consequently, defining RAS effector pathway(s) required for PDA initiation and maintenance is critical to improve treatment of this disease. Here, we show that expression of BRAF(V600E), but not PIK3CA(H1047R), in the mouse pancreas leads to pancreatic intraepithelial neoplasia (PanIN) lesions. Moreover, concomitant expression of BRAF(V600E) and TP53(R270H) result in lethal PDA. We tested pharmacologic inhibitors of RAS effectors against multiple human PDA cell lines. Mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) inhibition was highly effective both in vivo and in vitro and was synergistic with AKT inhibition in most cell lines tested. We show that RAF→MEK→ERK signaling is central to the initiation and maintenance of PDA and to rational combination strategies in this disease. These results emphasize the value of leveraging multiple complementary experimental systems to prioritize pathways for effective intervention strategies in PDA. SIGNIFICANCE: PDA is diffi cult to treat, in large part, due to recurrent mutations in the KRAS gene. Here, we defi ne rational treatment approaches for the disease achievable today with existing drug combinations by thorough genetic and pharmacologic dissection of the major KRAS effector pathways, RAF→MEK→ERK and phosphoinositide 3'-kinase (PI3'K)→AKT.

MEK1/2 inhibition elicits regression of autochthonous lung tumors induced by KRASG12D or BRAFV600E.

Genetically engineered mouse (GEM) models of lung tumorigenesis allow careful evaluation of lung tumor initiation, progression, and response to therapy. Using GEM models of oncogene-induced lung cancer, we show the striking similarity of the earliest stages of tumorigenesis induced by KRAS(G12D) or BRAF(V600E). Cre-mediated expression of KRAS(G12D) or BRAF(V600E) in the lung epithelium of adult mice initially elicited benign lung tumors comprising cuboidal epithelial cells expressing markers of alveolar pneumocytes. Strikingly, in a head-to-head comparison, oncogenic BRAF(V600E) elicited many more such benign tumors and did so more rapidly than KRAS(G12D). However, despite differences in the efficiency of benign tumor induction, only mice with lung epithelium expression of KRAS(G12D) developed malignant non-small cell lung adenocarcinomas. Pharmacologic inhibition of mitogen-activated protein (MAP)-extracellular signal-regulated kinase (ERK) kinase (MEK)1/2 combined with in vivo imaging showed that initiation and maintenance of both BRAF(V600E)- or KRAS(G12D)-induced lung tumors was dependent on MEK→ERK signaling. Although the tumors dramatically regressed in response to MEK1/2 inhibition, they regrew following cessation of drug treatment. Together, our findings show that RAF→MEK→ERK signaling is both necessary and sufficient for KRAS(G12D)-induced benign lung tumorigenesis in GEM models. The data also emphasize the ability of KRAS(G12D) to promote malignant lung cancer progression compared with oncogenic BRAF(V600E).