Molecular pharmacology and antitumor activity of Zalypsis in several human cancer cell lines.

Zalypsis is a new synthetic alkaloid tetrahydroisoquinoline antibiotic that has a reactive carbinolamine group. This functionality can lead to the formation of a covalent bond with the amino group of selected guanines in the DNA double helix, both in the absence and in the presence of methylated cytosines. The resulting complex is additionally stabilized by the establishment of one or more hydrogen bonds with adjacent nucleotides in the opposite strand as well as by van der Waals interactions within the minor groove. Fluorescence-based thermal denaturation experiments demonstrated that the most favorable DNA triplets for covalent adduct formation are AGG, GGC, AGC, CGG and TGG, and these preferences could be rationalized on the basis of molecular modeling results. Zalypsis-DNA adducts eventually give rise to double-strand breaks, triggering S-phase accumulation and apoptotic cell death. The potent cytotoxic activity of Zalypsis was ascertained in a 24 cell line panel. The mean IC(50) value was 7nM and leukemia and stomach tumor cell lines were amongst the most sensitive. Zalypsis administration in four murine xenograft models of human cancer demonstrates significant tumor growth inhibition that is highest in the Hs746t gastric cancer cell line with no weight loss of treated animals. Taken together, these results indicate that the potent antitumor activity of Zalypsis supports its current development in the clinic as an anticancer agent.

Activation of phosphatidylinositol 3-kinase by membrane localization of p110alpha predisposes mammary glands to neoplastic transformation.

Phosphatidylinositol 3-kinases (PI3K) constitute important regulators of various signaling pathways with relevance in cancer. Enhanced activation of p110alpha, the catalytic subunit of PI3K, was found in a high proportion of many human tumor types. We generated a mouse model in which PI3K is activated by forced recruitment of p110alpha to the membrane. Different transgenic lines expressing myristoylated p110alpha protein under the control of the epithelial-specific mouse mammary tumor virus promoter were selected according to different levels of PI3K activity and characterized. Delayed mammary gland involution and morphologic changes of the mammary ducts could be detected in young transgenic female mice. These changes were more pronounced in old animals, especially in mutiparous females, in which we observed increased ductal branching, alveolar hyperplasia, and intraductal neoplasia. We also observed a small percentage of mammary tumors. Crosses of myrp110alpha transgenic mice with heterozygous p53(+/-) knockout mice resulted in neither enhanced tumorigenesis nor in a stronger mammary gland phenotype. However, the CDK4 activating mutation (R24C) lead to increased tumorigenesis in transgenic myrp110alpha mice, emphasizing the postulated perturbation of the interaction of the CDK4/Rb/E2F cascade and the PI3K signaling in many human cancers. Interestingly, in tumors of myrp110alpha transgenic mice, we observed an increased phosphorylation of the estrogen receptor-alpha, a typical feature of human breast cancer. The model presented here will help to discover additional factors which influence the progression of preneoplastic lesions to tumors in the mammary gland and to explore antitumor therapies based on PI3K or estrogen receptor-alpha pathway inhibition.

Characterization of the p53 response to oncogene-induced senescence.

BACKGROUND: P53 activation can trigger various outcomes, among them reversible growth arrest or cellular senescence. It is a live debate whether these outcomes are influenced by quantitative or qualitative mechanisms. Furthermore, the relative contribution of p53 to Ras-induced senescence is also matter of controversy. METHODOLOGY/PRINCIPAL FINDINGS: This study compared situations in which different signals drove senescence with increasing levels of p53 activation. The study revealed that the levels of p53 activation do not determine the outcome of the response. This is further confirmed by the clustering of transcriptional patterns into two broad groups: p53-activated or p53-inactivated, i.e., growth and cellular arrest/senescence. Furthermore, while p53-dependent transcription decreases after 24 hrs in the presence of active p53, senescence continues. Maintaining cells in the arrested state for long periods does not switch reversible arrest to cellular senescence. Together, these data suggest that a Ras-dependent, p53-independent, second signal is necessary to induce senescence. This study tested whether PPP1CA (the catalytic subunit of PP1alpha), recently identified as contributing to Ras-induced senescence, might be this second signal. PPP1CA is induced by Ras; its inactivation inhibits Ras-induced senescence, presumably by inhibiting pRb dephosphorylation. Finally, PPP1CA seems to strongly co-localize with pRb only during senescence. CONCLUSIONS: The levels of p53 activation do not determine the outcome of the response. Rather, p53 activity seems to act as a necessary but not sufficient condition for senescence to arise. Maintaining cells in the arrested state for long periods does not switch reversible arrest to cellular senescence. PPP1CA is induced by Ras; its inactivation inhibits Ras-induced senescence, presumably by inhibiting pRb dephosphorylation. Finally, PPP1CA seems to strongly co-localize with pRb only during senescence, suggesting that PP1alpha activation during senescence may be the second signal contributing to the irreversibility of the senescent phenotype.

Loss-of-function genetic screening identifies a cluster of ribosomal proteins regulating p53 function.

Introduction of conditional murine p53 (p53val135) and oncogenic ras into double p53/p21-null mouse embryonic fibroblasts (MEFs) showed that p21waf1 was not required for combined ras/p53-induced senescent-like growth arrest. We used this cellular system to identify key players in the ras-p53-induced senescence in the absence of p21. Applying a retroviral-based genetic screen, we obtained mRNA antisense fragments against a cluster of 14 different ribosomal proteins which loss of function bypasses p53-induced growth arrest. The expression of the ribosomal protein antisense fragments reduced the transcriptional activity of p53. Experiments with eGFP-p53 chimeras suggest that the effect is mediated by a reduction of p53. To study whether p53 was downregulated by MDM2-dependent degradation, we tested the effect of the RP antisenses in double p53/MDM2-null MEFs and observed that in the absence of MDM2, reduction of the RP levels also decreases p53 levels. Therefore, although we cannot discard other unknown mechanism, we suggest that the decrease in the levels of ribosomal proteins might inhibit p53-specific translation. Finally, quantitative analysis comparing levels of mRNA in tumours versus mRNA in normal tissue of the same organ and patient showed that a variable percentage of lung, prostate or colon tumours have reduced levels of the RPs tested. Interestingly, in most cases, the reduction of ribosomal protein mRNAs occurs only to 50%. Our data suggest that ribosomal protein imbalance might contribute to p53 regulation through the ribosomal biogenesis checkpoint.

The PTEN/PI3K/AKT signalling pathway in cancer, therapeutic implications.

PTEN/PI3K/AKT constitutes an important pathway regulating the signaling of multiple biological processes such as apoptosis, metabolism, cell proliferation and cell growth. PTEN is a dual protein/lipid phosphatase which main substrate is the phosphatidyl-inositol,3,4,5 triphosphate (PIP3), the product of PI3K. Increase in PIP3 recruits AKT to the membrane where it is activated by other kinases also dependent on PIP3. Many components of this pathway have been described as causal forces in cancer. PTEN activity is lost by mutations, deletions or promoter methylation silencing at high frequency in many primary and metastatic human cancers. Germ line mutations of PTEN are found in several familial cancer predisposition syndromes. Activating mutations which have been reported for PI3K and AKT, in tumours are able to confer tumourigenic properties in several cellular systems. Additionally, the binding of PI3K to oncogenic ras is essential for the transforming properties of ras. In summary, the data strongly support the view of the PTEN/PI3K/AKT pathway as an important target for drug discovery.

PPP1CA contributes to the senescence program induced by oncogenic Ras.

Ectopic expression of conditional murine p53 (p53val135) and oncogenic ras is enough to induce a senescent-like growth arrest at the restrictive temperature. We took advantage of this cellular system to identify new key players in the ras/p53-induced senescence. Applying a retroviral-based genetic screen, we obtained an antisense RNA fragment against PPP1CA, the catalytic subunit of protein phosphatase 1alpha, whose loss of function bypasses ras/p53-induced growth arrest and senescence. Expression of a specific short hairpin (sh)RNA against PPP1CA impairs the p53-dependent induction of p21 after DNA damage and blocks the subsequent pRb dephosphorylation, thus bypassing p53-induced arrest. We found that oncogenic ras promotes an increase in the intracellular level of ceramides together with an increase in the PPP1CA protein levels. Addition of soluble ceramide to the cells induced a senescence phenotype that is blocked through PPP1CA downregulation by specific shRNA. Analysis of human tumors suggests that one of the PPP1CA alleles might be lost in a high percentage of carcinomas such as kidney and colorectal. The overexpression of two out of five PPP1CA alternative spliced variants reduced tumor cell growth and the downregulation of the protein to hemizygosity increased the anchorage-independent growth. We propose that oncogenic stress induced by ras causes ceramide accumulation, therefore, increasing PPP1CA activity, pRb dephosphorylation and onset of the p53-induced arrest, contributing to tumor suppression.

MAP17 inhibits Myc-induced apoptosis through PI3K/AKT pathway activation.

MAP17 is a non-glycosylated membrane-associated protein that has been shown to be over-expressed in human carcinomas, suggesting a possible role of this protein in tumorigenesis. However, very little is known about the molecular mechanism mediating the possible tumor promoting properties of MAP17. To analyze the effect of MAP17 on cell survival, we used Rat1 fibroblasts model where Myc over-expression promotes apoptosis in low serum conditions. In the present work, we report that over-expression of MAP17 protects Rat1a fibroblasts from Myc-induced apoptosis through reactive oxygen species (ROS)-mediated activation of the PI3K/AKT signaling pathway. MAP17-mediated survival was associated with absence of Bax translocation to the mitochondria and reduced caspase-3 activation. We show that a fraction of PTEN undergoes oxidation in MAP17-over-expressing cells. Furthermore, activation of AKT by MAP17 as measured by Thr308 phosphorylation was independent of PI3K activity. Importantly, modulation of ROS by antioxidant treatment prevented activation of AKT, restoring the level of apoptosis in serum-starved Rat1/c-Myc fibroblasts. Finally, over-expression of a dominant-negative mutant of AKT in MAP17-expressing clones makes them sensitive to serum depletion. Our data indicate that MAP17 protein activates AKT through ROS and this is determinant to confer resistance to Myc-induced apoptosis in the absence of serum.

MAP17 enhances the malignant behavior of tumor cells through ROS increase.

Tumorigenesis occurs when the mechanisms involved in the control of tissue homeostasis are disrupted and cells stop responding to physiological signals. Therefore, genes capable of desensitizing tumoral cells from physiological signals may provide a selective advantage within the tumoral mass and influence the outcome of the disease. We undertook a large-scale genetic screen to identify genes able to alter the cellular response to physiological signals and provide selective advantage once tumorigenesis has begun. We identified MAP17, a small 17 kDa non-glycosylated membrane protein previously identified by differential display being over-expressed in carcinomas. Tumor cells that over-express MAP17 show an increased tumoral phenotype with enhanced proliferative capabilities both in presence or absence of contact inhibition, decreased apoptotic sensitivity and increased migration. MAP17-expressing clones also grow better in nude mice. The increased malignant cell behavior induced by MAP17 are associated with an increase in reactive oxygen species (ROS) production, and the treatment of MAP17-expressing cells with antioxidants results in a reduction in the tumorigenic properties of these cells. Treatment of melanoma cells with inhibitors of Na+-coupled co-transporters lead to an inhibition of ROS increase and a decrease in the malignant cell behavior in MAP17-expressing clones. Finally, we show that MAP17-dependent ROS increase and tumorigenesis are dependent on its PDZ-binding domain, since disruption of its sequence by point mutations abolishes its ability to enhance ROS production and tumorigenesis. Our work shows the tumorigenic capability of MAP17 through a connection between Na+-coupled co-transporters and ROS.

MAP17 overexpression is a common characteristic of carcinomas.

We undertook a large-scale genetic screen to identify genes able to alter the cellular response to physiological signals and provide selective advantage once tumorigenesis has begun. We identified MAP17, a small 17 kDa non-glycosylated membrane protein previously identified, being overexpressed in carcinomas. We found that MAP17 is overexpressed in a great variety of human carcinomas. Immunohistochemical analysis of MAP17 during cancer progression shows, at least in prostate and ovarian carcinomas, that overexpression of the protein strongly correlates with tumoral progression (P < 0.0001). Many tumor cells also express MAP17 and its expression does not correlate with expression of SCL, a neighbor gene reported to be co-expressed in some hematopoietic cell lines. SCL neither is expressed in most MAP17-positive tumors, indicating the independent transcription of MAP17, at least in carcinomas. We cloned 5' genomic region to MAP17 and described the minimal promoter necessary to produce independent activation of MAP17. Moreover, we have found that MAP17 promoter is activated by oncogenes. Taken together, our data show an independent activation of MAP17 promoter that can be driven by oncogenes and that might explain the common overexpression of MAP17 in human carcinomas.

Levels of p27(kip1) determine Aplidin sensitivity.

Aplidin (plitidepsin) is a novel anticancer drug isolated from the marine tunicate Aplidium albicans. Aplidin shows potent antitumor activity in preclinical models against a wide variety of human tumors. Aplidin is currently in phase II clinical trials in a variety of solid tumors and hematologic malignancies. Moreover, clinical studies of Aplidin in combination with other agents are ongoing because it generally lacks cross-resistance with other known cytotoxic drugs. The mode of action of Aplidin in tumor cells is only partially understood. Aplidin induces an early oxidative stress response, which results in a rapid and sustained activation of the epidermal growth factor receptor, the nonreceptor protein tyrosine kinase Src, and the serine threonine kinases c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase. Here, we show that sensitivity to Aplidin correlates inversely with the levels of expression of the cyclin-dependent kinase inhibitor p27(kip1) (p27) in a panel of low passaged human sarcoma cell lines. Aplidin induces p27 through an oxidation-dependent mechanism and the reduction of p27 levels by specific short hairpin RNA increases Aplidin sensitivity. We confirmed these results in p27 null mouse embryonic fibroblasts corroborating the specificity of the p27 role in Aplidin response because p21(waf1) null mouse embryonic fibroblasts do not show this increased sensitivity. We propose a mechanism of action of Aplidin involving p27 and support the analysis of p27 in the response to Aplidin in currently ongoing clinical trials to establish the levels of this protein as response predictor.

PTEN, more than the AKT pathway.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/phosphatidylinositol 3-kinase (PI3K)/AKT constitute an important pathway regulating the signaling of multiple biological processes such as apoptosis, metabolism, cell proliferation and cell growth. PTEN is a dual protein/lipid phosphatase and its main substrate phosphatidyl-inositol 3,4,5 triphosphate (PIP3) is the product of PI3K. Increase in PIP3 recruits AKT to the membrane where is activated by other kinases also dependent on PIP3. Many components of this pathway have been described as causal forces in cancer. PTEN activity is lost by mutations, deletions or promoter methylation silencing at high frequency in many primary and metastatic human cancers. Germ line mutations of PTEN are found in several familial cancer predisposition syndromes. Recently, many activating mutations in the PI3KCA gene (coding for the p110alpha catalytic subunit of PI3K) have been described in human tumors. Activation of PI3K and AKT are reported to occur in breast, ovarian, pancreatic, esophageal and other cancers. Genetically modified mice confirm these PTEN activities. Tissue-specific deletions of PTEN usually provoke cancer. Moreover, an absence of PTEN cooperates with an absence of p53 to promote cancer. However, we have observed very different results with the expression of activated versions of AKT in several tissues. Activated AKT transgenic lines do not develop tumors in breast or prostate tissues and do not cooperate with an absence of p53. This data suggest that an AKT-independent mechanism contributes to PTEN tumorigenesis. Crosses with transgenic mice expressing possible PTEN targets indicate that neither cyclin D1 nor p53 are these AKT-independent targets. However, AKT is more than a passive bridge toward PTEN tumorigenesis, since its expression not only allows but also enforces and accelerates the tumorigenic process in combination with other oncogenes.

Mst1, RanBP2 and eIF4G are new markers for in vivo PI3K activation in murine and human prostate.

Phosphatidylinositol 3-kinases (PI3Ks) constitute important regulators of signaling pathways. The PIK3CA gene encoding the p110-alpha catalytic subunit represents one of the highly mutated oncogenes identified in human cancer. Here, we report new markers for in vivo PI3K activation in prostate. To that end, we used a transgenic mouse line, which expresses a constitutively active p110-alpha subunit in the epithelial cells of the prostate. The activity of the PI3K pathway in the prostate was proven by assessing the phosphorylation of the PI3K direct target AKT1 and of the mTOR target eukaryotic translation initiation factor 4G (eIF4G). To establish also transcriptional ('late') targets of the PI3K pathway, we tested two genes, Mst1 and RanBP2, which we recently described as transcriptional targets of the growth factor platelet-derived growth factor-beta. We show that the levels of both proteins are elevated in transgenic animals. Additionally, we describe that the phosphorylation of AKT and eIF4G, as well as the elevation of the Mst1 and RanBP2 protein levels, can be inhibited in vivo in transgenic animals by the PI3K inhibitor LY294002. Finally, we performed human tissue microarray experiments with the four markers. Since they define overlapping but not identical subsets of the tested tissue panel, a combination of all four markers might lead to a more accurate diagnosis of the status of the PI3K-signaling cascade in cancer patients.

Extreme sensitivity to Yondelis (Trabectedin, ET-743) in low passaged sarcoma cell lines correlates with mutated p53.

Yondelis (Trabectedin, ET-743) is a marine anticancer agent currently in Phase II/III development in patients with advanced pretreated soft tissue sarcoma. In the present study, we generated a panel of low passaged tumor cell lines from samples explanted from chemonaive sarcoma patients with different tumor types. We assessed in vitro sensitivity/resistance to Trabectedin and doxorubicin in a panel of sarcoma cell lines and examined the correlation between molecular alterations in DNA repair genes and sensitivity to Trabectedin. We treated cell lines with Trabectedin and doxorubicin in both 96-h and clonogenic assays. In both assays, well-defined groups of resistant and sensitive cell lines were observed. Resistance to Trabectedin did not correlate with resistance to doxorubicin, indicating that the two drugs may have different mechanisms of resistance. p53 mutations and deletions correlated with extreme sensitivity (IC50 < 1 nM) to Trabectedin (P < 0.01). In a pair of isogenic cell lines differing only in the presence or absence of wild-type p53, the absence of p53 rendered cells threefold more sensitive to Trabectedin.

Mice expressing myrAKT1 in the mammary gland develop carcinogen-induced ER-positive mammary tumors that mimic human breast cancer.

AKT1/PKB is a serine/threonine protein kinase that regulates biological processes such as proliferation, apoptosis and growth in a variety of cell types. To assess the oncogenic capability of an activated form of AKT in vivo we have generated several transgenic mouse lines that overexpress in the mammary epithelium the murine Akt1 gene modified with a myristoylation signal, which renders active this protein by localizing it to the plasma membrane. We demonstrate that expression of myristoylated AKT in the mammary glands increases the susceptibility of these mice to the induction of mammary tumors of epithelial origin by the carcinogen 9,10-dimethyl-1,2 benzanthracene (DMBA). We have found that while carcinogen-treated wild-type mice show mostly mammary tumors of sarcomatous origin, AKT transgenic mice treated with DMBA developed mainly adenocarcinoma or adenosquamous tumors, all of them displaying activated AKT. We analyzed other possible molecular alterations cooperating with AKT and found that neither Ras nor beta-catenin/Wnt pathways seemed altered nor p53 mutated. We have found that 100% of mammary DMBA-induced tumors and benign lesions in myrAKT mice are estrogen receptor (ERalpha)-positive and are more frequent than in wild-type littermates. These data show that AKT activation cooperates with deregulation of the estrogen receptor in the DMBA-induced mammary tumorigenesis model and recapitulate two characteristics of some human breast tumors. Thus, our model might provide a preclinical relevant model system to study the role of AKT and ERalpha in breast tumorigenesis and the response of mammary gland tumors to chemotherapeutics.

Inhibition of phosphatidylinositol-3-kinase synergizes with gemcitabine in low-passage tumor cell lines correlating with Bax translocation to the mitochondria.

Apoptotic pathways, including the phosphatidylinositol-3-kinase (PI3K)/AKT survival pathway, are altered in most cancer cells in relation to their normal counterparts and these differences may present an excellent therapeutic window. To gain insight into the relevance of the PI3K pathway as a target for drug discovery we generated tumor cell lines from different tumor samples that we maintained at low passage. The characterization of these cell lines indicates that all of them have constitutively activated the PI3K pathway through different mechanisms. All cell lines were differentially sensitive to the PI3K inhibitor LY294002. Our data also support previous work indicating that PI3K inhibition might help classical chemotherapeutic treatments such as gemcitabine and strengthen suboptimal doses that might be effective for these purposes in decreasing the risk of side-effects. Finally, the analysis of the molecular markers that might be implicated in the synergism between LY294002 and gemcitabine suggests that PI3K inhibition might aid chemotherapeutic treatment, leading to changes in the balance between anti- and pro-apoptotic molecules of the Bcl-2 family, Bcl-XL and Bax. These results facilitate the exploration of potential synergism between chemotherapeutic treatment and the search for others that can account for similar molecular mechanisms of cooperation.