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Summary: Although thousands of solid tumors have been sequenced to date, a fundamental under-sampling bias is inherent in current methodologies. This is caused by a tissue sample input of fixed dimensions (e.g., 6 mm biopsy), which becomes grossly under-powered as tumor volume scales. Here, we demonstrate representative sequencing (Rep-Seq) as a new method to achieve unbiased tumor tissue sampling. Rep-Seq uses fixed residual tumor material, which is homogenized and subjected to next-generation sequencing. Analysis of intratumor tumor mutation burden (TMB) variability shows a high level of misclassification using current single-biopsy methods, with 20% of lung and 52% of bladder tumors having at least one biopsy with high TMB but low clonal TMB overall. Misclassification rates by contrast are reduced to 2% (lung) and 4% (bladder) when a more representative sampling methodology is used. Rep-Seq offers an improved sampling protocol for tumor profiling, with significant potential for improved clinical utility and more accurate deconvolution of clonal structure.

Summary: The cell of origin of pancreatic ductal adenocarcinoma (PDAC) has been controversial. Here, we show that identical oncogenic drivers trigger PDAC originating from both ductal and acinar cells with similar histology but with distinct pathophysiology and marker expression dependent on cell of origin. Whereas acinar-derived tumors exhibited low AGR2 expression and were preceded by pancreatic intraepithelial neoplasias (PanINs), duct-derived tumors displayed high AGR2 and developed independently of a PanIN stage via non-mucinous lesions. Using orthotopic transplantation and chimera experiments, we demonstrate that PanIN-like lesions can be induced by PDAC as bystanders in adjacent healthy tissues, explaining the co-existence of mucinous and non-mucinous lesions and highlighting the need to distinguish between true precursor PanINs and PanIN-like bystander lesions. Our results suggest AGR2 as a tool to stratify PDAC according to cell of origin, highlight that not all PanIN-like lesions are precursors of PDAC, and add an alternative progression route to the current model of PDAC development. : Pancreatic intraepithelial neoplasias (PanINs) are established precursor lesions to pancreatic ductal adenocarcinoma (PDAC). Ferreira et al. show that this model represents acinar-derived PDAC and that, in contrast, duct-derived PDAC does not develop via PanINs. PanIN-like lesions occurring alongside duct-derived tumors arise from adjacent non-mutant tissue and do not represent tumor precursors. Keywords: PDAC, pancreatic cancer, mouse models, progression, PanINs, AGR2, cell of origin

Summary: RAS family GTPases contribute directly to the regulation of type I phosphoinositide 3-kinases (PI3Ks) via RAS-binding domains in the PI3K catalytic p110 subunits. Disruption of this domain of p110α impairs RAS-mutant-oncogene-driven tumor formation and maintenance. Here, we test the effect of blocking the interaction of RAS with p110α on epidermal growth factor receptor (EGFR)-mutant-driven lung tumorigenesis. Disrupting the RAS-PI3K interaction inhibits activation of both AKT and RAC1 in EGFR-mutant lung cancer cells, leading to reduced growth and survival, and inhibits EGFR-mutant-induced tumor onset and promotes major regression of established tumors in an autochthonous mouse model of EGFR-mutant-induced lung adenocarcinoma. The RAS-PI3K interaction is thus an important signaling node and potential therapeutic target in EGFR-mutant lung cancer, even though RAS oncogenes are not themselves mutated in this setting, suggesting different strategies for tackling tyrosine kinase inhibitor resistance in lung cancer. : The interaction between RAS and PI 3-kinase is essential for RAS-mutant-induced carcinogenesis. Murillo et al. show that in EGFR-mutant-driven lung cancer, disruption of the interaction of PI 3-kinase with normal RAS proteins blocks tumor initiation and promotes regression of existing tumors, highlighting an unexpected vulnerability of EGFR-driven lung cancer. Keywords: RAS, KRAS, EGFR, PI3K, PIK3CA, lung cancer, RAC1

In animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality.

Squamous cell carcinoma (SCC) can progress to malignant metastatic cancer, including an aggressive subtype known as spindle cell carcinoma (spSCC). spSCC formation involves epithelial-to-mesenchymal transition (EMT), yet the molecular basis of this event remains unknown. The transcriptional co-activator YAP undergoes recurrent amplification in human SCC and overexpression of YAP drives SCC formation in mice. Here, we show that human spSCC tumours also feature strong nuclear localisation of YAP and overexpression of activated YAP (NLS-YAP-5SA) with Keratin-5 (K5-CreERt) is sufficient to induce rapid formation of both SCC and spSCC in mice. spSCC tumours arise at sites of epithelial scratch wounding, where tumour-initiating epithelial cells undergo EMT to generate spSCC. Expression of the EMT transcription factor ZEB1 arises upon wounding and is a defining characteristic of spSCC in mice and humans. Thus, the wound healing response synergises with YAP to drive metaplastic transformation of SCC to spSCC.

Despite multiple reports of overexpression in prostate cancer (PC), the reliance of PC cells on activated epidermal growth factor receptor (EGFR) and its downstream signaling to phosphoinositide 3'-kinase/Akt (PI3K/Akt/PTEN) and/or mitogen-activated protein kinase (MAPK/ERK) pathways has not been fully elucidated. In this study, we compared the role of EGF-mediated signaling in nonmalignant (BPH-1, PNT1A, and PNT1 B) and PC cell lines (DU145, PC3, LNCaP, and CWR22Rv1). EGF-induced proliferation was observed in all EGFR-expressing PC cells except PC3, indicating that EGFR expression does not unequivocally trigger proliferation following EGF stimulation. ErbB2 recruitment potentiated EGF-induced signals and was associated with the most pronounced effects of EGF despite low EGFR expression. In this way, the sum of EGFR and ErbB2 receptor phosphorylation proved to be a more sensitive indicator of EGF-induced proliferation than quantification of the expression of either receptor alone. Both Akt and ERK were rapidly phosphorylated in response to EGF, with ERK phosphorylation being weakest in PC3 cells. Extrapolation of these findings to clinical PC suggests that assessment of phosphorylated EGFR + ErbB2 together could serve as a marker for sensitivity to anti-EGFR-targeted therapies.

In prostate cancer (PC), increasing evidence suggests that androgen receptor (AR) signalling is functional under conditions of maximal androgen blockade. PC cells survive and proliferate in the altered hormonal environment possibly by interactions between growth factor-activated pathways and AR signalling. The present review article summarizes the current evidence of this crosstalk and focuses on the interactions among the ErbB receptor network, its downstream pathways, the AR. The potential role of this crosstalk in the development of androgen independence and in relation to antiandrogen therapy is discussed. Such interactions provide insight into possible complementary or additional strategies in the management of PC.

MUC1 mucin is transcriptionally regulated by estrogen, progesterone, and glucocorticoids. Our objective was to determine whether androgen receptor. (20AR) activation regulates expression of MUC1. The following breast and prostatic cell lines were phenotyped and grouped according to AR and MUC1protein expression: 1) AR+MUCi + [DAR17+19. (20AR transfectants of DU-145), ZR-75-1, MDA-MB-453, and T47D]; 2) AR-MUCi+ [DZeoi. (20AR- vector control), DU-145, BT20, MDA-MB231, and MCF7]; 3) AIR +MUCi -. (20LNCaP and LNCaP-r). Cell proliferation was determined using the MTT assay in the presence of synthetic androgen R1881, 0.1 pM to 1 µM. Cell surface MUC1expression was determined by flow cytometry in the presence or absence of oestradiol, medroxy progesterone acetate or R1881, with and without 4 hydroxy-flutamide. (204-OH), a nonsteroidal AR antagonist. The functional significance of MUC1expression was investigated with a cell-cell aggregation assay. Only AR+ MUC1 + cell lines showed a significant increase in MUC1expression with AR activation. (20P. (20range) =.01 to .0001), reversed in the presence of 4-OHF. Cell proliferation was unaffected. Increased expression of MUC1was associated with a significant. (20P. (20range) =.002 to .001) reduction in cell-cell adhesion. To our knowledge, this is the first description of androgen-dependent regulation of MUC1mucin. This is also functionally associated with decreased cell-cell adhesion, a recognised feature of progressive malignancy. These findings have important implications for physiological and pathological processes.

Many common diseases of the gas exchange surface of the lung have no specific treatment but cause serious morbidity and mortality. Idiopathic Pulmonary Fibrosis (IPF) is characterized by alveolar epithelial cell injury, interstitial inflammation, fibroblast proliferation and collagen accumulation within the lung parenchyma. Keratinocyte Growth Factor (KGF, also known as FGF-7) is a critical mediator of pulmonary epithelial repair through stimulation of epithelial cell proliferation. During repair, the lung not only uses resident cells after injury but also recruits circulating bone marrow-derived cells (BMDC). Several groups have used Mesenchymal Stromal Cells (MSCs) as therapeutic vectors, but little is known about the potential of Hematopoietic Stem cells (HSCs). Using an inducible lentiviral vector (Tet-On) expressing KGF, we were able to efficiently transduce both MSCs and HSCs, and demonstrated that KGF expression is induced in a regulated manner both in vitro and in vivo. We used the in vivo bleomycin-induced lung fibrosis model to assess the potential therapeutic effect of MSCs and HSCs. While both populations reduced the collagen accumulation associated with bleomycin-induced lung fibrosis, only transplantation of transduced HSCs greatly attenuated the histological damage. Using double immunohistochemistry, we show that the reduced lung damage likely occurs through endogenous type II pneumocyte proliferation induced by KGF. Taken together, our data indicates that bone marrow transplantation of lentivirus-transduced HSCs can attenuate lung damage, and shows for the first time the potential of using an inducible Tet-On system for cell based gene therapy in the lung.