Oncogenic KrasG12D Expression Research Articles

Zebrafish rhabdomyosarcoma reflects the developmental stage of oncogene expression during myogenesis

Rhabdomyosarcoma is a pediatric malignancy thought to arise from the uncontrolled proliferation of myogenic cells. Here, we have generated models of rhabdomyosarcoma in the zebrafish by inducing oncogenic KRAS(G12D) expression at different stages during muscle development. Several zebrafish promoters were used, including the cdh15 and rag2 promoters, which drive gene expression in early muscle progenitors, and the mylz2 promoter, which is expressed in differentiating myoblasts. The tumors that developed differed in their ability to recapitulate normal myogenesis. cdh15:KRAS(G12D) and rag2:KRAS(G12D) fish developed tumors that displayed an inability to complete muscle differentiation as determined by histological appearance and gene expression analyses. By contrast, mylz2:KRAS(G12D) tumors more closely resembled mature skeletal muscle and were most similar to well-differentiated human rhabdomyosarcoma in terms of gene expression. mylz2:KRAS(G12D) fish showed significantly improved survival compared with cdh15:KRAS(G12D) and rag2:KRAS(G12D) fish. Tumor-propagating activity was enriched in myf5-expressing cell populations within all of the tumor types. Our results demonstrate that oncogenic KRAS(G12D) expression at different stages during muscle development has profound effects on the ability of tumor cells to recapitulate normal myogenesis, altering the tumorigenic capability of these cells.

Abstract 5008: A morphologically distinct cancer initiating cell in human and murine PanIN and pancreatic cancer.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is highly lethal with a survival rate of less than 6 months for the majority of patients. In order to better understand its development we hypothesized that there is cellular heterogeneity in murine and human PDAC precursor lesions (PanINs). We utilized two independent models of murine PanIN (mPanIN) formation to drive oncogenic KrasG12D expression in the murine pancreas during development (KCPdx1) or specifically in adult acinar cells (KCiMist1). The mice were crossed to the mTmG reporter mouse to map the fate of cells expressing Kras (KCiMist1G) that are marked by expression of GFP. We used transmission electron microscopy on mPanIN-containing tissue to identify a morphologically distinct rare cell type present in both models of mPanIN. These cells morphologically resemble tuft cells, which are characterized by their brush-like morphology, including apically projecting microvillae and the expression of DCLK1 and AcTub. Using Flourescence Activated Cell Sorting Techniques (FACS), we were able to isolate DCLK1HI/GFP+ murine PanIN tuft cells expressed in the KCiMist1G model. We discovered that these cells have an increased ability to form PanIN spheres in vitro relative to GFP+ cells alone, indicative of a progenitor PanIN stem cell function. Using immunofluorescent labeling on a human PanIN (n=23) and metastatic PDAC (n=10) tissue arrays, we determined that in 15% of PanIN lesions, 13-30% of the cells have detectable levels of AcTub, with a primarily apical pattern of labeling. In contrast, over 80% of metastatic PDAC cells are AcTub+, but they no longer exhibit an apical pattern of labeling. Applying our cell surface FACS strategy to four different human pancreatic cancer cell lines and three early passage human xenografts, we determined that 2-5% of human pancreatic cancer cells have detectable levels of cell-surface AcTub and DCLK1. We have functionally analyzed the tumor-initiating capacity of cell surface AcTubHI cells using in vitro tumor sphere experiments. In this assay, cell surface AcTubHI cells show a statistically significant 100-fold increase in tumor sphere-forming capacity relative to cell surface AcTubLOW cells. We further evaluated the functional significance of cell surface AcTub by analyzing the in vivo tumor initiating capacities of cell surface AcTubHI and AcTubLOW cells from a human PDAC xenograft. Cell surface AcTubHI cells exhibited significantly increased tumor initiating frequency relative to the AcTubLOW population (**p<0.0000539). These data provide important new information regarding cellular heterogeneity in murine PanIN and human pancreatic cancer, and suggest that novel strategies targeting DCLK1 and AcTub may have potential therapeutic utility. Citation Format: Jennifer M. Bailey, Janivette Alsina, Zeshaan Rasheed, Ya-Yuan Fu, Florencia McAllister, Pankaj Pasricha, William Matsui, Anirban Maitra, Steven Leach. A morphologically distinct cancer initiating cell in human and murine PanIN and pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5008. doi:10.1158/1538-7445.AM2013-5008

Transforming Growth Factor-β Receptor Type II Deletion Increases the Multiplicity and Growth of K-ras–induced Lung Cancers

Transforming growth factor-β (TGF-β) is a pluripotent regulator of epithelial growth and homeostasis and functions as a tumor suppressor in numerous malignancies. TGF-β signaling defects are common in many malignancies, including non–small cell lung cancer (NSCLC); however, the mechanisms by which defective TGF-β signaling promotes lung cancer growth and development are relatively unknown. We generated a novel NSCLC mouse model by simultaneously activating oncogenic KrasG12D expression and deleting TGF-β receptor type II (TGF-βRII) in airway epithelial cells. Genetic manipulation was targeted to the airway by K5Cre*PR, which uses a keratin 5 (K5) promoter to direct expression of an RU486-inducible, Cre recombinase-progesterone receptor fusion protein to epithelial cells. K5Cre*PR.lox-stop-lox-KrasG12D.TGF-βRII flox/flox mice treated with tracheal RU486 develop multifocal NSCLC beginning at approximately 32 weeks of age. Interestingly, these animals develop both thyroid transcription factor (TTF)-positive adenocarcinomas and K5-positive squamous cell carcinomas. TGF-βRII deletion increases tumor multiplicity from 3.2 ± 1.7 to 49 ± 10 tumors per animal and increases average tumor size from 0.71 to 1.04 mm, compared with tumors generated in K5Cre*PR.LSL-KrasG12D mice (P < 0.05 for both comparisons). In addition, K5Cre*PR.LSL-KrasG12D.TGFβRII flox/flox mice have both increased serum and bronchoalveolar lavage (BAL) TGF-β1 levels as well as increased BAL macrophages. Mechanistically, TGF-βRII deletion increases the multiplicity and growth of K-ras–induced lung cancers through mechanisms involving increased proliferation and increased inflammation.

Constitutive K‐RasG12D Activation of ERK2 Specifically Regulates 3D Invasion of Human Pancreatic Cancer Cells via MMP‐1

Pancreatic ductal adenocarcinomas (PDAC) are highly invasive &amp; metastatic neoplasms unresponsive to current therapies. Overwhelmingly, PDAC harbors constitutive, oncogenic mutations in K‐RasG12D that exist prior to invasion. Histologic &amp; genetic analyses of human PDAC exhibit increased expression of ERK1/2 and pro‐invasive MMPs; indicators of poor prognosis. However, the distinct molecular mechanisms necessary for K‐Ras – ERK1/2 signaling &amp; its influence on MMP‐directed stromal invasion in primary human pancreatic ductal epithelial cells (PDECs) has yet to be elucidated in 3D. Expression of oncogenic K‐RasG12D alone in genetically‐defined PDECs reveals increased invadopodia &amp; epithelial‐to‐mesenchymal transition markers, but only when cultured in a 3D basement membrane (BM) model. Activation of ERK2, but not ERK1, occurs only in K‐RasG12D mutated PDECs cultured in 3D &amp; is necessary for invasion based upon pharmacologic &amp; shRNA inhibition. Increased invasion of K‐RasG12D PDECs through the BM is associated with a specific microarray gene signature &amp; induction of MMPs. Specifically, MMP‐1 RNA, protein, &amp; proteolysis are amplified in K‐RasG12D PDECs when assayed by RT‐PCR, ELISA, &amp; FRET. Importantly, MMP‐1 silencing mimics ERK2 inhibition &amp; disrupts vertical invasion. ERK2‐isoform &amp; MMP‐1 targeting in 3D are shown to be viable strategies to attenuate invasion of K‐RasG12D mutated human pancreatic cancer cells. GPB is funded by an F30 DK088402‐01.

Constitutive K-RasG12D Activation of ERK2 Specifically Regulates 3D Invasion of Human Pancreatic Cancer Cells via MMP-1

Pancreatic ductal adenocarcinomas (PDAC) are highly invasive and metastatic neoplasms commonly unresponsive to current drug therapy. Overwhelmingly, PDAC harbors early constitutive, oncogenic mutations in K-Ras(G12D) that exist prior to invasion. Histologic and genetic analyses of human PDAC biopsies also exhibit increased expression of extracellular signal-regulated kinase (ERK) 1/2 and proinvasive matrix metalloproteinases (MMP), indicators of poor prognosis. However, the distinct molecular mechanisms necessary for K-Ras/ERK1/2 signaling and its influence on MMP-directed stromal invasion in primary human pancreatic ductal epithelial cells (PDEC) have yet to be elucidated in three-dimensions. Expression of oncogenic K-Ras(G12D) alone in genetically defined PDECs reveals increased invadopodia and epithelial-to-mesenchymal transition markers, but only when cultured in a three-dimensional model incorporating a basement membrane analog. Activation of ERK2, but not ERK1, also occurs only in K-Ras(G12D)-mutated PDECs cultured in three-dimensions and is a necessary intracellular signaling event for invasion based upon pharmacologic and short hairpin RNA (shRNA) inhibition. Increased active invasion of K-Ras(G12D) PDECs through the basement membrane model is associated with a specific microarray gene expression signature and induction of MMP endopeptidases. Specifically, MMP-1 RNA, its secreted protein, and its proteolytic cleavage activity are amplified in K-Ras(G12D) PDECs when assayed by real-time quantitative PCR, ELISA, and fluorescence resonance energy transfer (FRET). Importantly, shRNA silencing of MMP-1 mimics ERK2 inhibition and disrupts active, vertical PDEC invasion. ERK2 isoform and MMP-1 targeting are shown to be viable strategies to attenuate invasion of K-Ras(G12D)-mutated human pancreatic cancer cells in a three-dimensional tumor microenvironment.

Either Kras activation or Pten loss similarly enhance the dominant-stable CTNNB1-induced genetic program to promote granulosa cell tumor development in the ovary and testis.

WNT, PI3K or RAS signaling pathways control specific stages of ovarian follicular development. To analyze the functional interactions of these pathways in granulosa cells during follicular development in vivo, we generated specific mutant mouse models. Stable activation of the WNT signaling effector beta-catenin (CTNNB1) in granulosa cells results in the formation of premalignant lesions that develop into granulosa cell tumors (GCTs) spontaneously later in life or following targeted deletion of the tumor suppressor gene Pten. Conversely, expression of oncogenic KRASG12D dramatically arrests proliferation, differentiation and apoptosis in granulosa cells, and consequently, small abnormal follicle-like structures devoid of oocytes accumulate in the ovary. Because of the potent anti-proliferative effects of KRASG12D in granulosa cells, we sought to determine if KRASG12D would block precancerous lesion and tumor formation in follicles of the CTNNB1 mutant mice. Unexpectedly, transgenic Ctnnb1;Kras mutant mice exhibited increased GC proliferation, decreased apoptosis and impaired differentiation and developed early-onset GCTs leading to premature death in a manner similar to the Ctnnb1;Pten mutant mice. Microarray and RT-PCR analyses revealed that gene regulatory processes induced by CTNNB1 were mostly enhanced by either KRAS activation or Pten loss in remarkably similar patterns and degree. The concomitant activation of CTNNB1 and KRAS in Sertoli cells also caused testicular granulosa cell tumors that showed gene expression patterns that partially overlapped those observed in GCTs of the ovary. Although the mutations analyzed herein have not yet been linked to adult GCTs in humans, 1) other components of these pathways may be altered or mutated, 2) these mutations may relate to juvenile GCTs or 3) they may occur in tumors of other tissues where CTNNB1 is mutated. Importantly, our results provide strong evidence that CTNNB1 is the driver in these contexts and that KRASG12D and Pten loss promote the program set in motion by the CTNNB1.

Use of chromosome engineering to model a segmental deletion of chromosome band 7q22 found in myeloid malignancies

AbstractMonosomy 7 and del(7q) are associated with adverse features in myeloid malignancies. A 2.5-Mb commonly deleted segment (CDS) of chromosome band 7q22 is implicated as harboring a myeloid tumor suppressor gene (TSG); however, molecular analysis of candidate TSGs has not uncovered loss of function. To determine whether haploinsufficiency for the 7q22 CDS contributes to myeloid leukemogenesis, we performed sequential gene targeting to flank a region of orthologous synteny on mouse chromosome band 5A3 with loxP sites. We then generated Mx1-Cre, 5A3fl mutant mice and deleted the targeted interval in vivo. Although excision was inefficient, we confirmed somatic deletion of the 5A3 CDS in the hematopoietic stem cell compartment. Mx1-Cre, 5A3fl mice show normal hematologic parameters and do not spontaneously develop myeloid malignancies. The 5A3fl deletion does not cooperate with oncogenic KrasG12D expression, Nf1 inactivation, or retroviral mutagenesis to accelerate leukemia development and did not modulate responsiveness to antileukemia drugs. These studies demonstrate that it is feasible to somatically delete a large chromosomal segment implicated in tumor suppression in hematopoietic cell populations in vivo; however, our data do not support the hypothesis that the 7q22/5A3 CDS interval contains a myeloid TSG.

Abstract 4178: N-cadherin haploinsufficiency prolongs survival in a genetically engineered mouse model of pancreatic cancer

Abstract Cadherin switching (E-cadherin to N-cadherin) associated with epithelial-mesenchymal transition is implicated in the transition from benign tumors to invasive, malignant cancer and the subsequent metastatic dissemination of tumor cells. Although N-cadherin upregulation has been observed in many cancer types including pancreatic ductal adenocarcinoma (PDA), the importance of this cell adhesion receptor in disease progression is not well understood. The concomitant expression of oncogenic KrasG12D and a mutant Trp53R172H allele in the developing murine pancreas results in metastatic PDA that recapitulates the cognate features of human pancreatic cancer providing an excellent animal model to identify genetic changes required for tumor progression. Here we determine the consequences of genetically blocking the switch to N-cadherin in a mouse model of PDA. Two independent N-cadherin mutant alleles were used in these genetic studies. Remarkably, pancreatic cancer mice heterozygous for N-cadherin (n=17) survived 25% longer (177 vs. 142 days, p &amp;lt;0.05) than N-cadherin wild-type mice (n=16). The survival benefit may be due, at least in part, to increased cell death in the N-cadherin heterozygous tumors. Furthermore, cell death was also increased in tumor cell lines isolated from the N-cadherin heterozygous tumors demonstrating a cell-autonomous apoptotic phenotype. Interestingly, tumors lacking N-cadherin (i.e. null) did not show a survival benefit possibly due to compensation by an alternative metastatic promoting molecule(s). In conclusion, our genetic studies indicate that interfering with N-cadherin-mediated adhesion/signaling delays tumor progression, at least in part, by promoting tumor cell death, thus providing further evidence that N-cadherin antagonists may have therapeutic potential for the treatment of pancreatic cancer in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4178.

Oncogenic Kras and Notch-1 cooperate in T-cell acute lymphoblastic leukemia/lymphoma

Evaluation of: Kindler T, Cornejo MG, Scholl C et al. K-RasG12D-induced T-cell lymphoblastic lymphoma/leukemias harbor Notch-1 mutations and are sensitive to γ-secretase inhibitors. Blood 15, 112(8), 3373–3382 (2008).Mutations of the Ras family are one of the most common somatic events found in all human cancers, although they are relatively rare in T-cell acute lymphoblastic leukemia (T-ALL). In mice, conditional expression of oncogenic KrasG12D from its endogenous promoter causes a fatal myeloproliferative disorder, and only rarely a T-ALL-like disease. In the article being evaluated, the authors demonstrate that primary mice expressing oncogenic Kras have a block in T-cell differentiation at the double-negative 1 stage. Interestingly, most secondarily transplanted mice develop a fatal T-ALL-like disease. Sequencing of NOTCH-1 showed that 50% of these mice harbored truncating mutations in the PEST domain that would be predicted to activate Notch signaling. Cell lines established from some of the mice demonstrated sensitivity to γ-secretase inhibition, suggesting that even when NOTCH-1 mutations occur as secondary collaborating events, tumors retain a dependency on this pathway that might be exploitable clinically.

A Cre-loxP based mouse model for conditional somatic gene expression and knock-down in vivo using avian retroviral vectors

Site and time-specific somatic gene transfer using the avian sarcoma-leucosis retrovirus RCAS, has been shown to be a powerful tool to analyze gene function in vivo. RCAS retroviruses transduce only mammalian cells genetically engineered to express the avian retroviral receptor, TVA. Here, we have generated a knock-in mouse line termed LSL-R26Tva-lacZ with concomitant conditional expression of TVA and lacZ by targeting the Rosa26 locus. A loxP flanked transcriptional stop cassette was used for conditional activation of TVA and LacZ expression in a Cre recombinase dependent manner. To demonstrate the ability of this system for site and time-specific somatic gene transfer in vivo, we directed TVA expression to the pancreas. Introduction of an RCAS vector carrying an oncogenic KrasG12D expression cassette induced focal ductal pancreatic lesions that recapitulate human pancreatic intraepithelial neoplasias which progress to pancreatic ductal adenocarcinomas. TVA mediated infection of genetically engineered mice with endogenous expression of KrasG12D in pancreatic progenitor cells using RCAS virus carrying a short hairpin RNA directed against murine TP53, resulted in dramatically enhanced progression to invasive adenocarcinomas. These results show that conditional expression of TVA enables spatio-temporal gene expression and knock-down in a small subset of somatic cells in vivo. Therefore, it closely models carcinogenesis in humans where tumors evolve from somatic gene mutations in developmental normal cells. Combined with the growing number of Cre expression models, RCAS-TVA based gene expression and knock-down systems open up promising perspectives for analysis of gene function in a time-controlled and tissue-specific fashion in vitro and in vivo.

Expressing N-RasG12D from the Endogenous Promoter Induces Myeloproliferative Disease (MPD) and Cooperates with Retroviral Insertional Mutagenesis (RIM) To Generate Acute Myeloid Leukemia (AML).

Somatic NRAS and KRAS mutations are common in myeloid malignancies, particularly juvenile and adult chronic myelomonocytic leukemia (JMML and CMML) and AML. Use of the interferon-inducible Mx1-Cre transgenic strain to activate oncogenic KrasG12Dexpression from the endogenous Kras promoter induces an aggressive MPD with 100% penetrance that is fatal by ∼100 days of age. These Mx1-Cre, KrasG12Dmice do not develop AML, which suggests that additional mutations are required. To identify genes that might cooperate with KrasG12Din leukemogenesis, we performed RIM with MOL4070LTR virus. Mx1-Cre, KrasG12Dpups were injected with this virus at birth and with pIpC at 3 weeks of age to induce Cre recombinase expression. Approximately 12% of these mice developed either AML or a transplantable MPD-like leukemia. In parallel studies, we intercrossed Mx1-Cre mice with a novel strain of loxP-STOP-loxP (LSL) NrasG12D“knock in” mice to investigate the effects of expressing NrasG12Din the same experimental system. Although Mx1-Cre, NrasG12Dmice develop MPD, this disease is remarkably less aggressive with a much later onset of disease and longer survival compared to Kras mutant mice. Consistent with the attenuated disease phenotype, myeloid progenitors from Mx1-Cre, NrasG12Dmice form fewer cytokine independent colonies in methylcellulose cultures and are less hypersensitive to growth factors than KrasG12Dprogenitors. Importantly, 90% of Mx1-Cre, NrasG12Dmice that were injected with MOL4070LTR at birth and with pIpC at 3 weeks old developed AML or MPD-like leukemia, most of which demonstrate myelomonocytic features that are reminiscent of human M4 AML. We are cloning retroviral integrations from these leukemias and have identified Evi1 and other common insertions. Although both NRAS and KRAS are commonly mutated in myeloid malignancies, our data indicate that expressing these oncoproteins at endogenous levels in primary stem/progenitor cells results in distinct phenotypes. We recently showed that fetal liver cells infected with MSCV viruses expressing NrasG12D, Kras4aG12D, or Kras4bG12Ddemonstrate similar patterns of cytokine-independent colony formation and growth factor hypersensitivity. These data raise the possibility that variable levels of expression of Ras isoforms in different hematopoietic compartments may underlie some of our results. These models provide tractable systems for identifying cooperating genes in leukemogenesis and platforms for testing and designing new therapeutic agents.

Multiparameter Flow Cytometric Analysis Reveals Aberrant Phosphorylation of a Network of Effector Molecules in Defined Populations of Kras Mutant Bone Marrow.

Ras proteins regulate cell fates by cycling between an inactive, GDP-bound and an active, GTP-bound state. Ras activation is controlled by the opposing effects of guanine nucleotide exchange factors, which promote GTP binding, and GTPase activating proteins (GAPs), which markedly accelerate the slow intrinsic Ras GTPase. The mutant RAS alleles found in leukemia and other cancers encode proteins that accumulate in the GTP-bound conformation due to defective intrinsic GTPase activity and resistance to GAPs. These mutant Ras proteins, in turn, aberrantly activate effector pathways such as Raf/MEK/ERK, PI3 kinase/Akt, and Ral-GDS. To investigate the mechanisms by which hyperactive Ras deregulates myeloid growth, we previously generated Mx1-Cre, LSL-KrasG12D mice, which harbor a conditional oncogenic KrasG12D allele and uniformly develop a fatal myeloproliferative disease (MPD) upon induction of KrasG12D expression (Braun et al., PNAS 10, 597, 2004). This robust in vivo phenotype is associated with hypersensitivity of myeloid progenitors to cytokines in methylcellulose assays and elevated levels of Ras-GTP. However, we surprisingly found no increase in basal or growth factor-induced levels of phosphorylated MEK, ERK, and Akt in bone marrow cells from KrasG12D mice with MPD. To investigate this apparent paradox, we analyzed the activation states of MEK, ERK, Akt, and STAT5 under various conditions. By increasing the stringency of serum starvation prior to stimulation with GM-CSF, serum, or both, we uncovered reproducible hyperphosphorylation of MEK, Akt, and STAT5 in KrasG12D marrow. We observed similar phosphorylation patterns in marrow from mice one week after activation of oncogenic KrasG12D expression and from mice with advanced MPD, suggesting that myeloid lineage cells do not adapt to oncogenic Kras by modulating activation of downstream effectors on this time scale. Finally, we used multiparameter flow cytometric analysis of intracellular phosphoproteins to directly assay Ras effector pathways in defined populations of primary cells. We demonstrated that the c-kit-positive, lineage-negative (c-kit+/lin−) fraction contains the colony-forming unit granulocyte-macrophage (CFU-GM) progenitors in control and KrasG12D marrow and that the GM-CSF hypersensitivity characteristic of KrasG12D marrow is retained in this fraction. This population comprises 0.2–0.9% of nucleated marrow cells, making analysis by traditional methods difficult. Cells in this population exhibited distinct patterns of ERK and STAT5 phosphorylation in comparison with whole bone marrow. Importantly, c-kit+/lin− cells from KrasG12D mice demonstrated marked hyperphosphorylation of multiple signal transduction molecules under both starved and stimulated conditions. We conclude that KrasG12D expression from the endogenous Kras locus deregulates a network of signaling molecules in hematopoietic cells. This is most evident in a defined subset of progenitors that exhibits aberrant proliferation and differentiation in vitro. Multiparameter flow cytometry is a robust methodology for analyzing the activation status of signaling networks in primary cells that can be used to monitor responses to targeted therapeutics in disease-relevant populations of cells.