Oncogenic Ras/Her-2 mediate hyperproliferation of polarized epithelial cells in 3D cultures and rapid tumor growth via the PI3K pathway (original) (raw)
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RAS Interaction with PI3K: More Than Just Another Effector Pathway
2011
RAS proteins are small GTPases known for their involvement in oncogenesis: around 25% of human tumors present mutations in a member of this family. RAS operates in a complex signaling network with multiple activators and effectors, which allows them to regulate many cellular functions such as cell proliferation, differentiation, apoptosis, and senescence. Phosphatidylinositol 3-kinase (PI3K) is one of the main effector pathways of RAS, regulating cell growth, cell cycle entry, cell survival, cytoskeleton reorganization, and metabolism. However, it is the involvement of this pathway in human tumors that has attracted most attention. PI3K has proven to be necessary for RAS-induced transformation in vitro, and more importantly, mice with mutations in the PI3K catalytic subunit p110α that block its ability to interact with RAS are highly resistant to endogenous oncogenic KRASinduced lung tumorigenesis and HRAS-induced skin carcinogenesis. These animals also have a delayed development of the lymphatic vasculature. Many PI3K inhibitors have been developed that are now in clinical trials. However, it is a complex pathway with many feedback loops, and interactions with other pathways make the results of its inhibition hard to predict. Combined therapy with another RAS-regulated pathway such as RAF/MEK/ ERK may be the most effective way to treat cancer, at least in animal models mimicking the human disease. In this review, we will summarize current knowledge about how RAS regulates one of its best-known effectors, PI3K.
Signaling pathways in Ras-mediated tumorigenicity and metastasis
Proceedings of the National Academy of Sciences, 1998
The effector domain mutants of oncogenic Ras, V12S35 Ras, V12G37 Ras, and V12C40 Ras were tested for their abilities to mediate tumorigenic and metastatic phenotypes in athymic nude mice when expressed in NIH 3T3 fibroblasts. All mutants displayed comparable tumorigenic properties, but only the mutant that activates the Rafmitogen-activated protein kinase kinase (MEK)-extracellular regulated kinase (ERK) 1͞2 pathway, V12S35 Ras, induced tumors in the experimental metastasis assay. Furthermore, direct activation of the MEK-ERK1͞2 pathway in NIH 3T3 cells by mos or a constitutively active form of MEK was sufficient to induce metastasis whereas R-Ras, which fails to activate the ERK1͞2 pathway, is tumorigenic but nonmetastatic. The subcutaneous tumors and lung metastases derived from V12S35 Ras-transformed NIH 3T3 cells expressed higher levels of activated ERK1͞2 in culture when compared with the parental cellular pool before injection, indicating that selection for cells with higher levels of activated ERK1͞2 occurred during tumor growth and metastasis. By contrast, cells explanted from V12G37-Ras or V12C40-Ras-induced tumors did not show changes in the level of ERK1͞2 activation when compared with the parental cells. When tumorexplanted cell lines derived from each of the effector domain mutants were passaged one additional time in vivo, all mediated rapid tumor growth, but, again, only cells derived from V12S35 Ras-tumors formed numerous metastatic lesions within the lung. These results show that the metastatic properties of the Ras effector domain mutants segregate, and that, whereas Ras-mediated tumorigenicity can arise independently of ERK1͞2 activation, experimental metastasis appears to require constitutive activation of the ERK1͞2 pathway. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Cancer Research, 2013
The selective pressures leading to cancers with mutations in both KRAS and PIK3CA are unclear. Here, we show that somatic cell knockin of both KRAS G12V and oncogenic PIK3CA mutations in human breast epithelial cells results in cooperative activation of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in vitro, and leads to tumor formation in immunocompromised mice. Xenografts from double-knockin cells retain single copies of mutant KRAS and PIK3CA, suggesting that tumor formation does not require increased copy number of either oncogene, and these results were also observed in human colorectal cancer specimens. Mechanistically, the cooperativity between mutant KRAS and PIK3CA is mediated in part by Ras/p110α binding, as inactivating point mutations within the Ras-binding domain of PIK3CA significantly abates pathway signaling. In addition, Pdk1 activation of the downstream effector p90RSK is also increased by the combined presence of mutant ...
Oncogenesis, 2016
The gene encoding phosphatidylinositol 3-kinase catalytic subunit α-isoform (PIK3CA, p110α) is frequently activated by mutation in human cancers. Based on detection in some breast cancer precursors, PIK3CA mutations have been proposed to have a role in tumor initiation. To investigate this hypothesis, we generated a novel mouse model with a Cre-recombinase regulated allele of p110α (myristoylated-p110α, myr-p110α) along with p53 fl/fl deletion and Kras G12D also regulated by Cre-recombinase. After instillation of adenovirus-expressing Cre-recombinase into mammary ducts, we found that myr-p110α accelerated breast tumor initiation in a copy number-dependent manner. Breast tumors induced by p53 fl/fl ;Kras G12D with no or one copy of myr-p110α had predominantly sarcomatoid features, whereas two copies of myr-p110α resulted in tumors with a carcinoma phenotype. This novel model provides experimental support for importance of active p110α in breast tumor initiation, and shows that the amount of PI3K activity can affect the rate of tumor initiation and modify the histological phenotype of breast cancer.
Involvement of Ras activation in human breast cancer cell signaling, invasion, and anoikis
Cancer research, 2004
Although mutated forms of ras are not associated with the majority of breast cancers (<5%), there is considerable experimental evidence that hyperactive Ras can promote breast cancer growth and development. Therefore, we determined whether Ras and Ras-responsive signaling pathways were activated persistently in nine widely studied human breast cancer cell lines. Although only two of the lines harbor mutationally activated ras, we found that five of nine breast cancer cell lines showed elevated active Ras-GTP levels that may be due, in part, to HER2 activation. Unexpectedly, activation of two key Ras effector pathways, the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling pathways, was not always associated with Ras activation. Ras activation also did not correlate with invasion or the expression of proteins associated with tumor cell invasion (estrogen receptor ␣ and cyclooxygenase 2). We then examined the role of Ras signaling in mediating resistance to matrix deprivationinduced apoptosis (anoikis). Surprisingly, we found that ERK and phosphatidylinositol 3-kinase/AKT activation did not have significant roles in conferring anoikis resistance. Taken together, these observations show that Ras signaling exhibits significant cell context variations and that other effector pathways may be important for Ras-mediated oncogenesis, as well as for anoikis resistance, in breast cancer. Additionally, because ERK and AKT activation are not strictly associated with Ras activation, pharmacological inhibitors of these two signaling pathways may not be the best approach for inhibition of aberrant Ras function in breast cancer treatment.
Cancer Research, 2013
The selective pressures leading to cancers with mutations in both KRAS and PIK3CA are unclear. Here, we show that somatic cell knockin of both KRAS G12V and oncogenic PIK3CA mutations in human breast epithelial cells results in cooperative activation of the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in vitro, and leads to tumor formation in immunocompromised mice. Xenografts from doubleknockin cells retain single copies of mutant KRAS and PIK3CA, suggesting that tumor formation does not require increased copy number of either oncogene, and these results were also observed in human colorectal cancer specimens. Mechanistically, the cooperativity between mutant KRAS and PIK3CA is mediated in part by Ras/ p110a binding, as inactivating point mutations within the Ras-binding domain of PIK3CA significantly abates pathway signaling. In addition, Pdk1 activation of the downstream effector p90RSK is also increased by the combined presence of mutant KRAS and PIK3CA. These results provide new insights into mutant KRAS function and its role in carcinogenesis. Cancer Res; 73(11); 3248-61. Ó2013 AACR.
Molecular Cancer Research, 2012
Mutation of K-Ras is a frequent oncogenic event in human cancers, particularly cancers of lungs, pancreas, and colon. It remains unclear why some tissues are more susceptible to Ras-induced transformation than others. Here, we globally activated a mutant oncogenic K-Ras allele (K-Ras G12D ) in mice and examined the tissue-specific effects of this activation on cancer pathobiology, Ras signaling, tumor suppressor, DNA damage, and inflammatory responses. Within 5 to 6 weeks of oncogenic Ras activation, mice develop oral and gastric papillomas, lung adenomas, and hematopoietic hyperproliferation and turn moribund. The oral, gastric, and lung premalignant lesions display activated extracellular signal-regulated kinases (Erk)1/2 and NF-kB signaling as well as activated tumor suppressor and DNA damage responses. Other organs such as pancreas, liver, and small intestine do not exhibit neoplastic progression within 6 weeks following K-Ras G12D activation and do not show a potent tumor suppressor response. Even though robust Erk1/2 signaling is activated in all the tissues examined, the pErk1/2 distribution remains largely cytoplasmic in K-Ras G12D -refractory tissues (pancreas, liver, and intestines) as opposed to a predominantly nuclear localization in K-Ras G12D -induced neoplasms of lung, oral, and gastric mucosa. The downstream targets of Ras signaling, pElk-1 and c-Myc, are elevated in K-Ras G12D -induced neoplastic lesions but not in K-Ras G12D -refractory tissues. We propose that oncogenic K-Ras-refractory tissues delay oncogenic progression by spatially limiting the efficacy of Ras/Raf/Erk1/2 signaling, whereas K-Ras-responsive tissues exhibit activated Ras/Raf/Erk1/2 signaling, rapidly form premalignant tumors, and activate potent antitumor responses that effectively prevent further malignant progression. Mol Cancer Res; 10(6); 845-55. Ó2012 AACR.
RAS interaction with PI3K p110α is required for tumor-induced angiogenesis
Journal of Clinical Investigation, 2014
Class I PI3Ks are expressed in all mammalian cell types and consist of 4 di erent isoforms: p110 , p110 , p110 , and p110 . Class I PI3Ks share some common structural characteristics, such as a p85-binding domain (except p110 ), a C2 domain, a helical domain, and a RAS-binding domain (RBD). Previous results from our laboratory have shown that the PI3K p110 subunit (encoded by Pik3ca) is able to bind directly to RAS through the RBD (1, 2). In vitro work has shown that this interaction is required for proper signaling propagation through some receptor tyrosine kinases (RTKs), like EGFR or FGFR, and that its loss reduces cell growth . In vivo, the interaction of RAS with PI3K is also needed for proper development of embryonic lymphangiogenesis. Moreover, when the RBD of Pik3ca is mutated in such a way that RAS cannot bind to and activate PI3K, RAS-driven tumor development in the lung and skin is abrogated (5), and preexisting RAS-driven lung tumors undergo partial regression and long-term stasis (6).
Breast Cancer-Associated PIK3CA Mutations Are Oncogenic in Mammary Epithelial Cells
Cancer Research, 2005
Activation of the phosphoinositide 3-kinase (PI3K) pathway has been implicated in the pathogenesis of a variety of cancers. Recently, mutations in the gene encoding the p110A catalytic subunit of PI3K (PIK3CA) have been identified in several human cancers. The mutations primarily result in single amino acid substitutions, with >85% of the mutations in either exon 9 or 20. Multiple studies have shown that these mutations are observed in 18% to 40% of breast cancers. However, the phenotypic effects of these PIK3CA mutations have not been examined in breast epithelial cells. Herein, we examine the activity of the two most common variants, E545K and H1047R, in the MCF-10A immortalized breast epithelial cell line. Both variants display higher PI3K activity than wild-type p110A yet remain sensitive to pharmacologic PI3K inhibition. In addition, expression of p110A mutants in mammary epithelial cells induces multiple phenotypic alterations characteristic of breast tumor cells, including anchorage-independent proliferation in soft agar, growth factor-independent proliferation, and protection from anoikis. Expression of these mutant p110A isoforms also confers increased resistance to paclitaxel and induces abnormal mammary acinar morphogenesis in three-dimensional basement membrane cultures. Together, these data support the notion that the cancer-associated mutations in PIK3CA may significantly contribute to breast cancer pathogenesis and represent attractive targets for therapeutic inhibition. (Cancer Res 2005; 65(23): 10992-11000) Note: S. J. Isakoff and J.A. Engelman contributed equally to this work. Requests for reprints: Joan S. Brugge,