Jessica J. Gierut

K-Ras4A splice variant is widely expressed in cancer and uses a hybrid membrane-targeting motif

Significance The KRAS oncogene is mutated more frequently in human cancer than any other. The KRAS transcript is alternatively spliced to give rise to two products, K-Ras4A and K-Ras4B, both of which are oncogenic when KRAS is mutated. We detected significant amounts of each transcript in human tumor cells and colorectal carcinomas. We found that K-Ras4A is targeted to the plasma membrane by dual targeting motifs distinct from those of K-Ras4B. Because interfering with membrane association of Ras proteins remains one of the most attractive approaches to anti-Ras therapy, efforts in this direction will have to disrupt both the K-Ras4A and the K-Ras4B membrane-targeting pathways. The two products of the KRAS locus, K-Ras4A and K-Ras4B, are encoded by alternative fourth exons and therefore, possess distinct membrane-targeting sequences. The common activating mutations occur in exons 1 or 2 and therefore, render both splice variants oncogenic. K-Ras4A has been understudied, because it has been considered a minor splice variant. By priming off of the splice junction, we developed a quantitative RT-PCR assay for K-Ras4A and K-Ras4B message capable of measuring absolute amounts of the two transcripts. We found that K-Ras4A was widely expressed in 30 of 30 human cancer cell lines and amounts equal to K-Ras4B in 17 human colorectal tumors. Using splice variant-specific antibodies, we detected K-Ras4A protein in several tumor cell lines at a level equal to or greater than that of K-Ras4B. In addition to the CAAX motif, the C terminus of K-Ras4A contains a site of palmitoylation as well as a bipartite polybasic region. Although both were required for maximal efficiency, each of these could independently deliver K-Ras4A to the plasma membrane. Thus, among four Ras proteins, K-Ras4A is unique in possessing a dual membrane-targeting motif. We also found that, unlike K-Ras4B, K-Ras4A does not bind to the cytosolic chaperone δ-subunit of cGMP phosphodiesterase type 6 (PDE6δ). We conclude that efforts to develop anti–K-Ras drugs that interfere with membrane trafficking will have to take into account the distinct modes of targeting of the two K-Ras splice variants.

Mutant N-Ras protects colorectal cancer cells from stress-induced apoptosis and contributes to cancer development and progression

N-RAS is one member of a family of oncoproteins that are commonly mutated in cancer. Activating mutations in NRAS occur in a subset of colorectal cancers, but little is known about how the mutant protein contributes to the onset and progression of the disease. Using genetically engineered mice, we find that mutant N-RAS strongly promotes tumorigenesis in the context of inflammation. The protumorigenic nature of mutant N-RAS is related to its antiapoptotic function, which is mediated by activation of a noncanonical mitogen-activated protein kinase pathway that signals through STAT3. As a result, inhibition of MAP-ERK kinase selectively induces apoptosis in autochthonous colonic tumors expressing mutant N-RAS. The translational significance of this finding is highlighted by our observation that NRAS mutation correlates with a less favorable clinical outcome for patients with colorectal cancer. These data show for the first time the important role that N-RAS plays in colorectal cancer.

Proteomic analysis of pRb loss highlights a signature of decreased mitochondrial oxidative phosphorylation

The retinoblastoma tumor suppressor (pRb) protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb loss are largely unexplored. We acutely ablated Rb in adult mice and conducted a quantitative analysis of RNA and proteomic changes in the colon and lungs, where Rb(KO) was sufficient or insufficient to induce ectopic proliferation, respectively. As expected, Rb(KO) caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but, unexpectedly, their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb loss are coupled with proliferation but uncoupled from transcription. The proteomic changes in common between Rb(KO) tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and oxidative phosphorylation (OXPHOS) function. RB(KO) cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb loss altered mitochondrial pyruvate oxidation from (13)C-glucose through the TCA cycle in mouse tissues and cultured cells. Consequently, RB(KO) cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment.

Strategies to Achieve Conditional Gene Mutation in Mice

The laboratory mouse is an ideal model organism for studying disease because it is physiologically similar to human and also because its genome is readily manipulated. Genetic engineering allows researchers to introduce specific loss-of-function or gain-of-function mutations into genes and then to study the resulting phenotypes in an in vivo context. One drawback of using traditional transgenic and knockout mice to study human diseases is that many mutations passed through the germline can profoundly affect development, thus impeding the study of disease phenotypes in adults. New technology has made it possible to generate conditional mutations that can be introduced in a spatially and/or temporally restricted manner. Mouse strains carrying conditional mutations represent valuable experimental models for the study of human diseases and they can be used to develop strategies for prevention and treatment of these diseases. In this article, we will describe the most widely used DNA recombinase systems used to achieve conditional gene mutation in mouse models and discuss how these systems can be employed in vivo.

Network-level effects of kinase inhibitors modulate TNF-α–induced apoptosis in the intestinal epithelium

Network rewiring converts a survival-promoting kinase into a deadly one. Rewired for death The proinflammatory cytokine tumor necrosis factor–α (TNF-α) stimulates various kinases and can trigger cell death. Gierut et al. analyzed the kinetics of the phosphorylation of signaling proteins and the of induction of cell death in the intestinal epithelium of mice pretreated with various kinase inhibitors and then administered TNF-α. Unexpectedly, inhibitors that targeted the same kinase with equal efficiencies had different effects on TNF-α–induced apoptosis. For example, an inhibitor of a kinase enhanced cell death, whereas other inhibitors targeting this kinase reduced cell death. Mathematical analysis of the data predicted, and in vivo experiments confirmed, that the kinase Akt, which usually promotes cell survival, increased TNF-α–induced apoptosis when the network had been altered by the death-enhancing inhibitor. Together, these data illustrate that network context is just as important as drug target when predicting the response to an inhibitor. Individual signaling pathways operate in the context of the broader signaling network. Thus, the response of a cell to signals from the environment is affected by the state of the signaling network, such as the clinically relevant example of whether some components in the network are inhibited. The cytokine tumor necrosis factor–α (TNF-α) promotes opposing cellular behaviors under different conditions; the outcome is influenced by the state of the network. For example, in the mouse intestinal epithelium, inhibition of the mitogen-activated protein kinase (MAPK) kinase MEK alters the timing of TNF-α–induced apoptosis. We investigated whether MAPK signaling directly influences TNF-α–induced apoptosis or whether network-level effects secondary to inhibition of the MAPK pathway alter the cellular response. We found that inhibitors of the MAPK kinase kinase Raf, MEK, or extracellular signal–regulated kinase (ERK) exerted distinct effects on the timing and magnitude of TNF-α–induced apoptosis in the mouse intestine. Furthermore, even different MEK inhibitors exerted distinct effects; one, CH5126766, potentiated TNF-α–induced apoptosis, and the others reduced cell death. Computational modeling and experimental perturbation identified the kinase Akt as the primary signaling node that enhanced apoptosis in the context of TNF-α signaling in the presence of CH5126766. Our work emphasizes the importance of integrated network signaling in specifying cellular behavior in response to experimental or therapeutic manipulation. More broadly, this study highlighted the importance of considering the network-level effects of pathway inhibitors and showed the distinct effects of inhibitors that share the same target.

Whole-Mount X-Gal Staining of Mouse Tissues

Although the development of improved mouse models, including conditional deletions, marks an exciting time in mouse genetics, it is important to characterize and validate these models. Cre reporter strains allow researchers to assess the recombinase expression profile and function in individual Cre mouse lines. These strains are engineered to express a reporter gene (usually LacZ) following the removal of a floxed STOP cassette, thus marking cell lineages that can be targeted with a given Cre line. This protocol provides a detailed method for the histochemical detection of β-galactosidase activity in Cre mouse strains.

Producing and concentrating lenti-Cre for mouse infections.

Lentiviral vectors offer versatility as vehicles for gene delivery. They can transduce a wide range of cell types and integrate into the host genome, which results in long-term expression of the transgene (Cre) both in vitro and in vivo. This protocol describes how lentiviral particles are produced, purified, and concentrated.

In Vivo Delivery of Lenti-Cre or Adeno-Cre into Mice Using Intranasal Instillation

Lung cancer remains the leading cause of cancer deaths among both men and women, with a lower rate of survival than both breast and prostate cancer. Development of the Cre/lox system and improved mouse models have allowed researchers to gain a better understanding of human disease, including lung cancer. Through the viral delivery of Cre, gene function in adult mice can be precisely studied at a specific developmental stage or in a specific cell/tissue type of choice. This protocol describes how to produce adenovirus-Cre precipitate. Using this adeno-Cre (or lentivirus-Cre), Cre can be expressed in mouse lungs. The virus is delivered by intranasal instillation.

Oncogenic K-Ras promotes proliferation in quiescent intestinal stem cells ☆ ☆☆

K-Ras is a monomeric GTPase that controls cellular and tissue homeostasis. Prior studies demonstrated that mutationally activated K-Ras (K-Ras(G12D)) signals through MEK to promote expansion and hyperproliferation of the highly mitotically active transit-amplifying cells (TACs) in the intestinal crypt. Its effect on normally quiescent stem cells was unknown, however. Here, we have used an H2B-Egfp transgenic system to demonstrate that K-Ras(G12D) accelerates the proliferative kinetics of quiescent intestinal stem cells. As in the TAC compartment, the effect of mutant K-Ras on the quiescent stem cell is dependent upon activation of MEK. Mutant K-Ras is also able to increase self-renewal potential of intestinal stem cells following damage. These results demonstrate that mutant K-Ras can influence intestinal homeostasis on multiple levels.

Abstract B04: Activating K-RasA146T mutations induce Mapk-dependent hyperproliferation in the intestinal epithelium

Ras is the most commonly mutated oncogene in human cancer, and is highly mutated in pancreatic, lung, and colorectal cancer. The most commonly reported canonical activating point mutations occur in codons 12, 13, and 61. Colorectal cancer, in particular, harbors a number of noncanonical K-Ras mutations, including mutations at codon 146, which occur in ~4% of patients. To understand the biologic effects of K-Ras A146T expression in the intestinal epithelium and other tissues, we engineered a conditional Cre recombinase-dependent mutant allele ( K-Ras LSL-A146T ), expressed from the endogenous KRas locus. We crossed K-Ras LSL-A146T mice to Fabpl-Cre mice, which express Cre recombinase in the colonic and distal small intestinal epithelium, and compared the phenotype of Fabpl-Cre; K-Ras LSL-A146T/+ mice to that of Fabpl-Cre; K-Ras LSL-G12D/+ mice. Compared to K-Ras G12D , expression of K-Ras A146T results in a mild hyperplastic and hyperproliferative phenotype, while expression of K-Ras G12D causes drastic hyperplasia and hyperproliferation. Additionally, we found that K-Ras A146T expression activates Mapk signaling and that K-Ras A146T -induced hyperproliferation is Mapk-dependent. Interestingly, unlike Fabpl-Cre; K-Ras LSL-G12D mice, which lack Paneth cells in the small intestine, Fabpl-Cre; K-Ras LSL-A146T/+ mice have intact Paneth cells. Finally, we found that although K-Ras G12D expression in the pancreas causes neoplastic transformation, K-Ras A146T expression has no detectable effect on pancreatic homeostasis, even after one year. Together, our results suggest that compared to the canonical K-Ras G12D mutation, activating K-Ras A146T mutations have a similar but milder effect on colonic homeostasis. Additionally, consistent with human data, we found that K-Ras A146T mutations have tissue-specific effects on neoplastic transformation. Citation Format: Emily Poulin, Jessica Gierut, Kevin Haigis. Activating K-Ras A146T mutations induce Mapk-dependent hyperproliferation in the intestinal epithelium [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B04.