Journal: Genes & Development

Article Title: Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α

doi: 10.1101/gad.249409.114

Figure Lengend Snippet: PI3K/Akt pathway activation in pancreatic cancer and pharmacological inhibition of p110α catalytic activity prevent the development of pancreatic ADM lesions induced by injury in the context of mutated Kras. ( A ) Analysis of p110 isoforms and its regulatory subunit expression by Western blot in microdissected murine acinar cells ( left ; N = 4) or whole-pancreas lysates ( right ; N = 4). Total spleen was used as a control for p110δ and p110γ expression (isoforms overexpressed in immune cells). p85 and total Akt correspond to loading controls. ( B ) p110s and p85 expression in human samples. N = 3. ( C ) Levels of pAkt in human PDAC. Scale, 200 μm. ( D ) Scheme. ( E , F ) Quantification of ADM numbers/fields and edema histological score ( E ) and representative hematoxylin and eosin (H&E) and immunohistochemistry (IHC) stainings ( F ; insets are in high magnification) of young KC mice injected with caerulein in the presence of the specific p110α inhibitor A66 ( N = 3) or vehicle ( N = 4) and sacrificed 8 h post-injections. ADM numbers were analyzed on six random 20×-magnification fields per representative slide for each mouse; edema scoring was achieved on a representative slide for each mouse. Mean ± SEM; (**) P < 0.001, Student’s t -test. IHC using indicated antibodies. Quantification of nuclear pERK staining was performed on five 20× magnification fields in each mouse normalized with the total number of nuclei. Mean ± SEM; (NS) Student’s t -test. Scale, 500 or 90 μm.

Article Snippet: Reagents were purchased as follows: A66 (32 nM for p110α, >12,500 nM for p110β, >12,500 nM for p110δ, 3480 nM for p110γ, and >5000 nM for mTOR in vitro IC50) ( ) and TGX-221 (5000 nM for p110α, 5 nM for p110β, 100 nM for p110δ, and >10,000 nM for p110γ in vitro IC50) were from Axon Medchem, caerulein and dexamethasone were from Sigma, and EGF was from R&D Systems.

Techniques: Activation Assay, Inhibition, Activity Assay, Expressing, Western Blot, Control, Immunohistochemistry, Injection, Staining

Journal: Genes & Development

Article Title: Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α

doi: 10.1101/gad.249409.114

Figure Lengend Snippet: Genetic inactivation of p110α catalytic activity in the pancreas prevents the development of mutated Kras-induced pancreatic preneoplastic and neoplastic lesions. ( A ) Experimental setup to express kinase-dead p110α and oncogenic Kras G12D in the pancreas. ( B ) Survival curve. Expected Mendelian ratios for Cre + ;p110α lox/lox were found; total number of pups was 155. N > 10 per genotype. ( C ) Percentage of 6-mo-old mice harboring no lesions, ADM, or PanINs depending on genotypes. ( D ) Representative H&E stains ( insets show representative areas in high magnification; scale, 500 or 100 μm) and indicated IHC analysis on paraffin-embedded KC, KC;p110α +/lox , and KC;p110α lox/lox pancreata. (Arrowheads) Pancreatic lobules with stromal Ki67-positive cells present in areas of ADM ; (em) early metaplastic transitions; (*) ADM lesions; (#) low-grade PanIns; (red arrowheads) CK19-positive normal ducts. CK19 is a well-defined ductal cell marker overexpressed first at the basolateral membrane of acinar cells (em) that undergo ADM. N = 4. ( E ) Pancreatic sections of KC, KC;p110α +/lox , and KC;p110α lox/lox animals were analyzed for the presence of pS473Akt, pAkt substrate, and pThr202/Tyr204ERK1/2 (cytoplasmic and nuclear staining) by IHC on serial sections (4 μm). Slides were counterstained with hematoxylin. Representative pictures are shown ( N = 4). Scale, 100 μm. ( F ) Experimental setup to express kinase-dead p110β and oncogenic Kras G12D in the pancreas. ( G ) Percentage of 6-mo-old mice harboring no lesions, ADM, or PanINs depending on genotypes. ( H ) H&E.

Article Snippet: Reagents were purchased as follows: A66 (32 nM for p110α, >12,500 nM for p110β, >12,500 nM for p110δ, 3480 nM for p110γ, and >5000 nM for mTOR in vitro IC50) ( ) and TGX-221 (5000 nM for p110α, 5 nM for p110β, 100 nM for p110δ, and >10,000 nM for p110γ in vitro IC50) were from Axon Medchem, caerulein and dexamethasone were from Sigma, and EGF was from R&D Systems.

Techniques: Activity Assay, Marker, Membrane, Staining

Journal: Genes & Development

Article Title: Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α

doi: 10.1101/gad.249409.114

Figure Lengend Snippet: Acinar p110α activity is crucial for induction and maintenance of pancreatic ADM lesions by tissue injury, also in the genetic context of mutated Kras. ( A ) Experimental setup. ( B , C ) Representative H&E staining ( insets in high magnification) of caerulein- or vehicle-injected 8-wk-old wild-type (WT) or C;p110α lox/lox ( B ) and KC, KC;p110α +/lox or KC;p110α lox/lox ( C ) mice 1 or 5 d after the last injection of caerulein. Dashed lines in insets surround a mixed acinar–ductal metaplasic structure. (#) Normal acinar cells containing characteristic zymogen granules. Scale, 4–50–500 μm. ( D , E ) Quantification of ADM or PanINs per field (magnification, 20×; six random fields per mouse; normalization per field). Each point represents a single mouse, and horizontal bars represent the mean percentage for each group. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, Student’s t -test, KC;p110α +/lox or KC;p110α lox/lox versus KC. ( F ) Phase contrast images of Ptf1a Cre ; Kras G12D acinar cells 5 d after isolation and treatment with 50 ng/mL TGF-α in the presence of increasing doses of p110α-specific inhibitor A66 (1–10 μM) or vehicle. Quantification of the percentage of ductal structures after 5 d in culture. N = 3 ex vivo acinar cultures from independent mice; (*) P < 0.05; (**) P < 0.01, Student’s t -test). Scale, 50 μm. ( G ) Rat AR4-2J B13 acinar cells were subjected to DEX+EGF (1 μM + 20 ng/mL) with the indicated treatment for 5 d to induce ADM. Immunofluorescence staining was performed as indicated, and representative confocal images are presented ( N ≥ 2). Scale, 10 μm.

Article Snippet: Reagents were purchased as follows: A66 (32 nM for p110α, >12,500 nM for p110β, >12,500 nM for p110δ, 3480 nM for p110γ, and >5000 nM for mTOR in vitro IC50) ( ) and TGX-221 (5000 nM for p110α, 5 nM for p110β, 100 nM for p110δ, and >10,000 nM for p110γ in vitro IC50) were from Axon Medchem, caerulein and dexamethasone were from Sigma, and EGF was from R&D Systems.

Techniques: Activity Assay, Staining, Injection, Isolation, Ex Vivo, Immunofluorescence

Journal: Genes & Development

Article Title: Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α

doi: 10.1101/gad.249409.114

Figure Lengend Snippet: p110α activity stimulates in vivo actin cytoskeleton remodeling and activation of Rho small GTPases during pancreatic cancer initiation. ( A ) Confocal microscopic images of actin staining on paraffin-embedded pancreata in the indicated treatments and genotypes 1 d after the last caerulein injection ( N ≥ 3 per genotype). White arrowheads show cortical actin staining, and white asterisks indicate the lumen, the diameter of which is enlarged during acinar-to-ductal transition. Scale, 10 μm. ( B ) Representative scheme of actin staining during ADM. ( C ) F-actin staining of A66 (5 μM), TGX-221 (0.5 μM), or vehicle-treated AR4-2J B13 acinar cells that were transdifferentiated in “ductal-like” cells by DEX+EGF for 5 d. Scale, 10 μm. Intensity of signal quantified by epifluorescence on five fields per condition. N = 3; (***) P < 0.001, Student’s t -test. ( E , top panel) Western blot analysis of pancreatic lysates from KC compared with KC;p110α lox/lox or KC;p110β lox/lox mice ( N ≥ 3) sacrificed 1 d after the last caerulein (Cae.) or PBS injection. Relative quantification is shown in the bottom panel. N ≥ 3; (**) P < 0.01; (*) P < 0.05, Student’s t -test. ( F ) Rho and Rac1 in vivo activities from pancreatic lysates of the indicated genotypes 1 d after caerulein treatment. N ≥ 2; (*) P < 0.05, Student’s t -test. Positive and negative controls correspond to GTP and GDP loading, respectively (not shown). ( G ) Overexpression of constitutively active RhoA or wild-type (WT) RhoA in AR4-2J B13 acinar cells treated as indicated. Scale, 10 μm. Transduction of acinar cells with GFP-expressing vectors leads to a >95% rate of transduction and was similarly affected by treatments compared with wild-type AR4-2J cells (not shown). RhoA overexpression was verified by Western blot; p-Cofilin was used as a control of constitutive activity of RhoA (not shown).

Article Snippet: Reagents were purchased as follows: A66 (32 nM for p110α, >12,500 nM for p110β, >12,500 nM for p110δ, 3480 nM for p110γ, and >5000 nM for mTOR in vitro IC50) ( ) and TGX-221 (5000 nM for p110α, 5 nM for p110β, 100 nM for p110δ, and >10,000 nM for p110γ in vitro IC50) were from Axon Medchem, caerulein and dexamethasone were from Sigma, and EGF was from R&D Systems.

Techniques: Activity Assay, In Vivo, Activation Assay, Staining, Injection, Western Blot, Quantitative Proteomics, Over Expression, Transduction, Expressing, Control

Journal: Genes & Development

Article Title: Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α

doi: 10.1101/gad.249409.114

Figure Lengend Snippet: p110α activity triggers the maintenance of inflammatory and proliferative pathways in the pancreas. ( A ) Ras in vivo activity from pancreatic lysates of KC compared with KC;p110α lox/lox and KC;p110β lox/lox mice ( N ≥ 3) sacrificed 1 d after the last caerulein (Cae.) injection. The relative quantification of all mice analyzed is shown below. (*) P < 0.05, Student’s t -test. Positive and negative controls correspond to GTP and GDP loading of Raf1-RBD, respectively (data not shown). ( B ) Immunohistochemical analysis of p65, pY705STAT3, pERK1/2, IL6, and αSMA on KC and KC;p110α +/lox compared with KC;p110α lox/lox ( N = 3) pancreata 1 or 5 d after the last caerulein or vehicle injection. Black arrowheads show acinar (pERK, pSTAT3, or p65) or stromal (IL6 or αSMA) positively stained cells. Scale, 100 μm. ( C ) Western blot analysis for STAT3 phosphorylation and EGFR expression of pancreatic lysates from KC compared with KC;p110α lox/lox mice sacrificed 1 d after the last caerulein or PBS injection. ( D ) EGFR IHC as indicated. Arrowheads show membrane staining. Scale, 50 μm. N = 3. ( E ) IHC analysis of pS473Akt, pAkt substrate, and pERK1/2 in caerulein-injected KC mice sacrificed 4 mo after the last injection. Black arrowheads show positive ADM or PanIN structures as indicated. N = 3. Scale, 100 μm.

Article Snippet: Reagents were purchased as follows: A66 (32 nM for p110α, >12,500 nM for p110β, >12,500 nM for p110δ, 3480 nM for p110γ, and >5000 nM for mTOR in vitro IC50) ( ) and TGX-221 (5000 nM for p110α, 5 nM for p110β, 100 nM for p110δ, and >10,000 nM for p110γ in vitro IC50) were from Axon Medchem, caerulein and dexamethasone were from Sigma, and EGF was from R&D Systems.

Techniques: Activity Assay, In Vivo, Injection, Quantitative Proteomics, Immunohistochemical staining, Staining, Western Blot, Phospho-proteomics, Expressing, Membrane

Journal: Genes & Development

Article Title: Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α

doi: 10.1101/gad.249409.114

Figure Lengend Snippet: p110α activity is required for the transition from preneoplastic lesions to adenocarcinoma induced by oncogenic Kras and mutated p53. ( A ) Experimental setup. ( B ) Survival curves. N ≥ 6 for each genotype. ( C ) Percentage of 4.5-mo-old mice harboring no lesions, ADM/PanIN1, PanIN2, or high-grade PanIN3/PDAC pancreatic lesions depending on genotypes. ( D ) Quantification of total pancreatic lesions in Pdx1-Cre; LSL-Tp53 R172H ; LSL-Kras G12D (KPC; n = 9), KPC;p110α +/lox ( n = 8), and KPC;p110α lox/lox ( n = 8) mice. (*) P < 0.05, Mann-Whitney test. ( E ) Representative H&E stainings and IHC using the indicated antibodies ( insets show representative areas in high magnification) of KPC, KPC;p110α +/lox , and KPC;p110α lox/lox mice. Scale, 2–100–200 μm. ( F ) MEF cells were stably transfected with murine shp53 with or without Kras G12V . Cell proliferation during 4 d in the presence of A66 (10 μM) or vehicle was analyzed. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, two-way ANOVA.

Article Snippet: Reagents were purchased as follows: A66 (32 nM for p110α, >12,500 nM for p110β, >12,500 nM for p110δ, 3480 nM for p110γ, and >5000 nM for mTOR in vitro IC50) ( ) and TGX-221 (5000 nM for p110α, 5 nM for p110β, 100 nM for p110δ, and >10,000 nM for p110γ in vitro IC50) were from Axon Medchem, caerulein and dexamethasone were from Sigma, and EGF was from R&D Systems.

Techniques: Activity Assay, MANN-WHITNEY, Stable Transfection, Transfection

Journal: Genes & Development

Article Title: Pancreatic cell plasticity and cancer initiation induced by oncogenic Kras is completely dependent on wild-type PI 3-kinase p110α

doi: 10.1101/gad.249409.114

Figure Lengend Snippet: p110α activity is required for pancreatic cancerogenesis. Schematic representation. (Lof) Loss of function.

Article Snippet: Reagents were purchased as follows: A66 (32 nM for p110α, >12,500 nM for p110β, >12,500 nM for p110δ, 3480 nM for p110γ, and >5000 nM for mTOR in vitro IC50) ( ) and TGX-221 (5000 nM for p110α, 5 nM for p110β, 100 nM for p110δ, and >10,000 nM for p110γ in vitro IC50) were from Axon Medchem, caerulein and dexamethasone were from Sigma, and EGF was from R&D Systems.

Techniques: Activity Assay