Principles of K-Ras effector organization and the role of oncogenic K-Ras in cancer initiation through G1 cell cycle deregulation

Illustrated here is the critical role of oncogenic KRAS in the initiation of cancer through deregulation of the G1 cell cycle, and elements and scenarios taking place under physiological conditions and in KRAS-driven cancer. Raf, PI3K and RalGDS are major K-Ras effectors. They bind at the same Ras site. What decides the cell selection among them? This temporal and spatial decision is critical since in some cellular context the outcome of their signaling pathways may oppose each other. Key among them is the concentration of calcium/calmodulin, negative feedback loops, where a downstream member of the pathway inhibits its upstream activator and cross-inhibition, where inhibition entails blocking another pathway. These three elements, in addition to spatial restrictions by K-Ras-membrane interactions, are not independent; they integrate to provide blueprints for cell decisions. Importantly, elucidation of signaling requires not only K-Ras binary interactions; but the structures and dynamics of its multiprotein complexes.     

K-Ras~(G12D)基因突变体慢病毒载体的构建及其鉴定

目的:构建K-RasG12D基因突变体慢病毒载体。方法:从病人组织中提取RNA通过RT-PCR反转录获得cDNA作为K-RasG12D基因模板,通过PCR法扩增出K-RasG12D基因突变体片段。将酶切的片段克隆入真核表达载体pCDH-CMV-MCS-EF1-RFP中,构建K-RasG12D基因突变体逆转录病毒真核表达载体。将连接产物转化至感受态大肠埃希菌DH5α,挑取转化平板上的细菌克隆,在抗生素培养液中培养过夜后进行PCR鉴定。经测序正确后转染293T细胞系,利用重组质粒PCR及串联基因表达的检测等方法对目的基因的转录与表达进行分析与鉴定。结果:所构建的K-RasG12D突变体基因逆转录病毒真核表达载体经PCR鉴定和测序鉴定正确,转染293T细胞后可以观察到可检测到高强度表达的RFP荧光信号。结论:成功构建了重组真核表达载体,为下一步建立稳定转染细胞系及进一步研究K-Ras突变在癌症发病中的作用奠定了基础。     

Investigation of the structural requirements of K-Ras(G12D) selective inhibitory peptide KRpep-2d using alanine scans and cysteine bridging

A structure-activity relationship study of a K-Ras(G12D) selective inhibitory cyclic peptide, KRpep-2d was performed. Alanine scanning of KRpep-2d focusing on the cyclic moiety showed that Leu⁷, Ile⁹, and Asp¹² are the key elements for K-Ras(G12D) selective inhibition of KRpep-2d. The cysteine bridging was also examined to identify the stable analog of KRpep-2d under reductive conditions. As a result, the KRpep-2d analog (12) including mono-methylene bridging showed potent K-Ras(G12D) selective inhibition in both the presence and the absence of dithiothreitol. This means that mono-methylene bridging is an effective strategy to obtain a reduction-resistance analog of parent disulfide cyclic peptides. Peptide 12 inhibited proliferation of K-Ras(G12D)-driven cancer cells significantly. These results gave valuable information for further optimization of KRpep-2d to provide novel anti-cancer drug candidates targeting the K-Ras(G12D) mutant.     

Temporal dissection of K-ras(G12D) mutant in vitro and in vivo using a regulatable K-ras(G12D) mouse allele

Animal models which allow the temporal regulation of gene activities are valuable for dissecting gene function in tumorigenesis. Here we have constructed a conditional inducible estrogen receptor-K-ras(G12D) (ER-K-ras(G12D)) knock-in mice allele that allows us to temporally switch on or off the activity of K-ras oncogenic mutant through tamoxifen administration. In vitro studies using mice embryonic fibroblast (MEF) showed that a dose of tamoxifen at 0.05 μM works optimally for activation of ER-K-ras(G12D) independent of the gender status. Furthermore, tamoxifen-inducible activation of K-ras(G12D) promotes cell proliferation, anchor-independent growth, transformation as well as invasion, potentially via activation of downstream MAPK pathway and cell cycle progression. Continuous activation of K-ras(G12D) in vivo by tamoxifen treatment is sufficient to drive the neoplastic transformation of normal lung epithelial cells in mice. Tamoxifen withdrawal after the tumor formation results in apoptosis and tumor regression in mouse lungs. Taken together, these data have convincingly demonstrated that K-ras mutant is essential for neoplastic transformation and this animal model may provide an ideal platform for further detailed characterization of the role of K-ras oncogenic mutant during different stages of lung tumorigenesis.     

The ganglioside G(D2) induces the constitutive activation of c-Met in MDA-MB-231 breast cancer cells expressing the G(D3) synthase

We have recently established and characterized cellular clones deriving from MDA-MB-231 breast cancer cells that express the human G(D3) synthase (GD3S), the enzyme that controls the biosynthesis of b- and c-series gangliosides. The GD3S positive clones show a proliferative phenotype in the absence of serum or growth factors and an increased tumor growth in severe immunodeficient mice. This phenotype results from the constitutive activation of the receptor tyrosine kinase c-Met in spite of the absence of ligand and subsequent activation of mitogen-activated protein kinase/extracellular signal-regulated kinase and phosphoinositide 3-kinase/Akt pathways. Here, we show by mass spectrometry analysis of total glycosphingolipids that G(D3) and G(D2) are the main gangliosides expressed by the GD3S positive clones. Moreover, G(D2) colocalized with c-Met at the plasma membrane and small interfering RNA silencing of the G(M2)/G(D2) synthase efficiently reduced the expression of G(D2) as well as c-Met phosphorylation and reversed the proliferative phenotype. Competition assays using anti-G(D2) monoclonal antibodies also inhibit proliferation and c-Met phosphorylation of GD3S positive clones in serum-free conditions. Altogether, these results demonstrate the involvement of the disialoganglioside G(D2) in MDA-MB-231 cell proliferation via the constitutive activation of c-Met. The accumulation of G(D2) in c-Met expressing cells could therefore reinforce the tumorigenicity and aggressiveness of breast cancer tumors.     

D1 in G2

No Abstract Available

Key Words

D-type Cyclins, Cyclin D1, Ras     

The role of Shp2 (PTPN11) in cancer

Tyrosyl phosphorylation, which is controlled by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs), regulates numerous cellular processes. Altered expression and/or mutations in PTKs are linked to many forms of cancer, yet until recently little was known about the roles of PTPs in normal cells or in cancer. Earlier work established that a member of the PTP superfamily, PTEN, is an important tumor suppressor gene. We now know that at least one other PTP, the SH2 domain-containing phosphatase Shp2, is a bona fide oncogene that is mutated in several types of leukemia and hyperactivated by other mechanisms in some solid tumors. Understanding how Shp2 and other PTPs contribute to oncogenesis should provide new insights into pathogenesis and might suggest new targets for anti-neoplastic drugs.     

G1m(1) and G1m(2) allotypes in Albanian towns of Calabria

The G1m(1) and G1m(2) allotype distribution was analyzed in a population sample from 11 Albanian towns of Calabria. The unusually high frequency of the G1m(1) marker already observed in Calabria as well as the presence of the Gm(2) phenotype were shown. The Calabrian and Albanian populations were similar, but significantly different from other Italian populations.     

A role for the G12 family of heterotrimeric G proteins in prostate cancer invasion

Many studies have suggested a role for the members of the G12 family of heterotrimeric G proteins (Galpha12 and Galpha13) in oncogenesis and tumor cell growth. However, few studies have examined G12 signaling in actual human cancers. In this study, we examined the role of G12 signaling in prostate cancer. We found that expression of the G12 proteins is significantly elevated in prostate cancer. Interestingly, expression of the activated forms of Galpha12 or Galpha13 in the PC3 and DU145 prostate cancer cell lines did not promote cancer cell growth. Instead, expression of the activated forms of Galpha12 or Galpha13 in these cell lines induced cell invasion through the activation of the RhoA family of G proteins. Furthermore, inhibition of G12 signaling by expression of the RGS domain of the p115-Rho-specific guanine nucleotide exchange factor (p115-RGS) in the PC3 and DU145 cell lines did not reduce cancer cell growth. However, inhibition of G12 signaling with p115-RGS in these cell lines blocked thrombin- and thromboxane A2-stimulated cell invasion. These observations identify the G12 family proteins as important regulators of prostate cancer invasion and suggest that these proteins may be targeted to limit invasion- and metastasis-induced prostate cancer patient mortality.     

Distribution of G1m(1), G1m(2) and G3m(5) allotypes in Aragon.

The distribution of G1m(1), G1m(2) and G3m(5) allotypes was studied in 700 unrelated individuals from Aragon (North-East Spain). The Gm haplotype frequencies were similar to those reported in French areas next to Aragon.     

Identification of epidermal Pdx1 expression discloses different roles of Notch1 and Notch2 in murine Kras(G12D)-induced skin carcinogenesis in vivo

Background

The Ras and Notch signaling pathways are frequently activated during development to control many diverse cellular processes and are often dysregulated during tumorigenesis. To study the role of Notch and oncogenic Kras signaling in a progenitor cell population, Pdx1-Cre mice were utilized to generate conditional oncogenic Kras^G12D mice with ablation of Notch1 and/or Notch2.

Methodology/Principal Findings

Surprisingly, mice with activated Kras^G12D and Notch1 but not Notch2 ablation developed skin papillomas progressing to squamous cell carcinoma providing evidence for Pdx1 expression in the skin. Immunostaining and lineage tracing experiments indicate that PDX1 is present predominantly in the suprabasal layers of the epidermis and rarely in the basal layer. Further analysis of keratinocytes in vitro revealed differentiation-dependent expression of PDX1 in terminally differentiated keratinocytes. PDX1 expression was also increased during wound healing. Further analysis revealed that loss of Notch1 but not Notch2 is critical for skin tumor development. Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and β-catenin signaling inhibition capabilities.

Conclusions/Significance

Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function. In addition, this finding may be relevant for research using Pdx1-Cre transgenic strains. Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors.     

Tissue and serum microRNAs in the Kras(G12D) transgenic animal model and in patients with pancreatic cancer

microRNAs (miRs) modulate the expression levels of mRNAs and proteins and can thus contribute to cancer initiation and progression. In addition to their intracelluar function, miRs are released from cells and shed into the circulation. We postulated that circulating miRs could provide insight into pathways altered during cancer progression and may indicate responses to treatment. Here we focus on pancreatic cancer malignant progression. We report that changes in miR expression patterns during progression of normal tissues to invasive pancreatic adenocarcinoma in the p48-Cre/LSL-Kras(G12D) mouse model mirrors the miR changes observed in human pancreatic cancer tissues. miR-148a/b and miR-375 expression were found decreased whereas miR-10, miR-21, miR-100 and miR-155 were increased when comparing normal tissues, premalignant lesions and invasive carcinoma in the mouse model. Predicted target mRNAs FGFR1 (miR-10) and MLH1 (miR-155) were found downregulated. Quantitation of nine microRNAs in plasma samples from patients distinguished pancreatic cancers from other cancers as well as non-cancerous pancreatic disease. Finally, gemcitabine treatment of control animals and p48-Cre/LSL-Kras(G12D) animals with pancreatic cancer caused distinct and up to 60-fold changes in circulating miRs that indicate differential drug effects on normal and cancer tissues. These findings support the significance of detecting miRs in the circulation and suggests that circulating miRs could serve as indicators of drug response.     

The oncogenic role of GASC1 in chemically induced mouse skin cancer

Gene amplified in squamous cell carcinoma (SCC) 1 (GASC1), also known as KDM4C/JMJD2C, encodes a histone demethylase that specifically demethylates lysine residues (H3K9, H3K36, and H1.4K26) and plays a crucial role in the regulation of gene expression as well as in heterochromatin formation. GASC1 is located at human chromosome 9p23–24, where frequent genomic amplification is observed in human esophageal cancer, and its aberrant expression is detected in a variety of human cancers, such as breast, colon, and prostate. Therefore, it is highly likely that GASC1 contributes to the genesis and/or development of cancer. However, there is a lack of direct evidence of GASC1 having an oncogenic function. In this study, we aimed to clarify the role of GASC1 in the skin SCC carcinogenesis. For this purpose, we generated Gasc1-heterozygous mice (Gasc1 ^+/−) with reduced expression of Gasc1. On the basis of our results, Gasc1 ^+/− mice displayed a significantly lower incidence and multiplicity of both benign and malignant tumors induced by the two-stage skin carcinogenesis protocol than wild-type mice. In addition, the volume of carcinoma was significantly lower in Gasc1 ^+/− mice. Consistent with these observations, knocking down of Gasc1 resulted in reduced cell viability of SCC cells in vitro. Our findings clearly demonstrated that GASC1 has an oncogenic role in skin carcinogenesis.

     

PKCdelta and MAPK mediate G(1) arrest induced by PMA in SKBR-3 breast cancer cells

The effects of activating endogenous protein kinase C (PKC) on cell proliferation and the cell cycle were investigated by treating the breast cancer cell line SKBR-3 with phorbol 12-myristate 13 acetate (PMA). This inhibited cell growth in a concentration-dependent manner, causing a marked arrest of cells in G(1). Pre-treatment with GF109203X completely blocked the antiproliferative effect of PMA, and pre-treatment with the PKCdelta inhibitor rottlerin partially blocked it. Infecting SKBR-3 cells with an adenovirus vector containing wild-type PKCdelta, WTPKCdeltaAdV, had similar effects on PMA. Infecting the cells with a dominant-negative PKCdeltaAdV construct blocked the growth inhibition induced by PMA. Downstream of PKC, PMA treatment inhibited extracellular signal-regulated kinase mitogen-activated protein kinase phosphorylation, up-regulated c-jun NH(2)-terminal kinase phosphorylation, and inhibited retinoblastoma (Rb) phosphorylation. These results strongly implicated PKC (mainly PKCdelta) in the G(1) arrest induced by PMA and suggested PKC as a target for breast cancer treatment.     

The role of N-linked glycosylation in determining the surface expression,G protein interaction and effector coupling of the alpha (alpha) isoform of the human thromboxane A(2) receptor

In humans, thromboxane (TX) A(2) signals through two TXA(2) receptor (TP) isoforms, termed TPalpha and TPbeta, that diverge exclusively within the carboxyl terminal cytoplasmic domains. The amino terminal extracellular region of the TPs contains two highly conserved Asn (N)-linked glycosylation sites at Asn(4) and Asn(16). While it has been established that impairment of N-glycosylation of TPalpha significantly affects ligand binding/intracellular signalling, previous studies did not ascertain whether N-linked glycosylation was critical for ligand binding per se or whether it was required for the intracellular trafficking and the functional expression of TPalpha on the plasma membrane (PM). In the current study, we investigated the role of N-linked glycosylation in determining the functional expression of TPalpha, by assessment of its ligand binding, G protein coupling and intracellular signalling properties, correlating it with the level of antigenic TPalpha protein expressed on the PM and/or retained intracellularly. From our data, we conclude that N-glycosylation of either Asn(4) or Asn(16) is required and sufficient for expression of functionally active TPalpha on the PM while the fully non-glycosylated TPalpha(N4,N16-Q4,Q16) is almost completely retained within the endoplasmic reticulum (ER) and remains functionally inactive, failing to associate with its coupling G protein Galpha(q) and, in turn, failing to mediate phospholipase (PL) Cbeta activation.