Resistance to dual blockade of the kinases PI3K and mTOR in KRAS-mutant colorectal cancer models results in combined sensitivity to inhibition of the receptor tyrosine kinase EGFR.

Targeted blockade of aberrantly activated signaling pathways is an attractive therapeutic strategy for solid tumors, but drug resistance is common. KRAS is a frequently mutated gene in human cancer but remains a challenging clinical target. Inhibitors against KRAS signaling mediators, namely, PI3K (phosphatidylinositol 3-kinase) and mTOR (mechanistic target of rapamycin), have limited clinical efficacy as single agents in KRAS-mutant colorectal cancer (CRC). We investigated potential bypass mechanisms to PI3K/mTOR inhibition in KRAS-mutant CRC. Using genetically engineered mouse model cells that had acquired resistance to the dual PI3K/mTOR small-molecule inhibitor PF-04691502, we determined with chemical library screens that inhibitors of the ERBB [epidermal growth factor receptor (EGFR)] family restored the sensitivity to PF-04691502. Although EGFR inhibitors alone have limited efficacy in reducing KRAS-mutant tumors, we found that PF-04691502 induced the abundance, phosphorylation, and activity of EGFR, ERBB2, and ERBB3 through activation of FOXO3a (forkhead box O 3a), a transcription factor inhibited by the PI3K to AKT pathway. PF-04691502 also induced a stem cell-like gene expression signature. KRAS-mutant patient-derived xenografts from mice treated with PF-04691502 had a similar gene expression signature and exhibited increased EGFR activation, suggesting that this drug-induced resistance mechanism may occur in patients. Combination therapy with dacomitinib (a pan-ERBB inhibitor) restored sensitivity to PF-04691502 in drug-resistant cells in culture and induced tumor regression in drug-resistant allografts in mice. Our findings suggest that combining PI3K/mTOR and EGFR inhibitors may improve therapeutic outcome in patients with KRAS-mutant CRC.

Cross-species analysis of genetically engineered mouse models of MAPK-driven colorectal cancer identifies hallmarks of the human disease.

Effective treatment options for advanced colorectal cancer (CRC) are limited, survival rates are poor and this disease continues to be a leading cause of cancer-related deaths worldwide. Despite being a highly heterogeneous disease, a large subset of individuals with sporadic CRC typically harbor relatively few established 'driver' lesions. Here, we describe a collection of genetically engineered mouse models (GEMMs) of sporadic CRC that combine lesions frequently altered in human patients, including well-characterized tumor suppressors and activators of MAPK signaling. Primary tumors from these models were profiled, and individual GEMM tumors segregated into groups based on their genotypes. Unique allelic and genotypic expression signatures were generated from these GEMMs and applied to clinically annotated human CRC patient samples. We provide evidence that a Kras signature derived from these GEMMs is capable of distinguishing human tumors harboring KRAS mutation, and tracks with poor prognosis in two independent human patient cohorts. Furthermore, the analysis of a panel of human CRC cell lines suggests that high expression of the GEMM Kras signature correlates with sensitivity to targeted pathway inhibitors. Together, these findings implicate GEMMs as powerful preclinical tools with the capacity to recapitulate relevant human disease biology, and support the use of genetic signatures generated in these models to facilitate future drug discovery and validation efforts.

Combination PI3K/MEK inhibition promotes tumor apoptosis and regression in PIK3CA wild-type, KRAS mutant colorectal cancer.

PI3K inhibition in combination with other agents has not been studied in the context of PIK3CA wild-type, KRAS mutant cancer. In a screen of phospho-kinases, PI3K inhibition of KRAS mutant colorectal cancer cells activated the MAPK pathway. Combination PI3K/MEK inhibition with NVP-BKM120 and PD-0325901 induced tumor regression in a mouse model of PIK3CA wild-type, KRAS mutant colorectal cancer, which was mediated by inhibition of mTORC1, inhibition of MCL-1, and activation of BIM. These findings implicate mitochondrial-dependent apoptotic mechanisms as determinants for the efficacy of PI3K/MEK inhibition in the treatment of PIK3CA wild-type, KRAS mutant cancer.

Development of a colon cancer GEMM-derived orthotopic transplant model for drug discovery and validation.

PURPOSE: Effective therapies for KRAS-mutant colorectal cancer (CRC) are a critical unmet clinical need. Previously, we described genetically engineered mouse models (GEMM) for sporadic Kras-mutant and non-mutant CRC suitable for preclinical evaluation of experimental therapeutics. To accelerate drug discovery and validation, we sought to derive low-passage cell lines from GEMM Kras-mutant and wild-type tumors for in vitro screening and transplantation into the native colonic environment of immunocompetent mice for in vivo validation. EXPERIMENTAL DESIGN: Cell lines were derived from Kras-mutant and non-mutant GEMM tumors under defined media conditions. Growth kinetics, phosphoproteomes, transcriptomes, drug sensitivity, and metabolism were examined. Cell lines were implanted in mice and monitored for in vivo tumor analysis. RESULTS: Kras-mutant cell lines displayed increased proliferation, mitogen-activated protein kinase signaling, and phosphoinositide-3 kinase signaling. Microarray analysis identified significant overlap with human CRC-related gene signatures, including KRAS-mutant and metastatic CRC. Further analyses revealed enrichment for numerous disease-relevant biologic pathways, including glucose metabolism. Functional assessment in vitro and in vivo validated this finding and highlighted the dependence of Kras-mutant CRC on oncogenic signaling and on aerobic glycolysis. CONCLUSIONS: We have successfully characterized a novel GEMM-derived orthotopic transplant model of human KRAS-mutant CRC. This approach combines in vitro screening capability using low-passage cell lines that recapitulate human CRC and potential for rapid in vivo validation using cell line-derived tumors that develop in the colonic microenvironment of immunocompetent animals. Taken together, this platform is a clear advancement in preclinical CRC models for comprehensive drug discovery and validation efforts.

Protein disulfide isomerase-associated 6 is an ATF6-inducible ER stress response protein that protects cardiac myocytes from ischemia/reperfusion-mediated cell death.

Proper folding of secreted and transmembrane proteins made in the rough endoplasmic reticulum (ER) requires oxygen for disulfide bond formation. Accordingly, ischemia can impair ER protein folding and initiate the ER stress response, which we previously showed is activated in the ischemic heart and in culture cardiac myocytes subjected to simulated ischemia. ER stress and ischemia activate the transcription factor, activating transcription factor 6 (ATF6), which induces numerous genes, many of which have not been identified, or examined in the heart. Using an ATF6 transgenic mouse model, we previously showed that ATF6 protected the heart from ischemic damage; however, the mechanism of this protection remains to be determined. In this study, we showed that, in the mouse heart, and in cultured cardiac myocytes, ATF6 induced the protein disulfide isomerase associated 6 (PDIA6) gene, which encodes an ER enzyme that catalyzes protein disulfide bond formation. Moreover, in cultured cardiac myocytes, ER stress-mediated PDIA6 promoter activation was ATF6-dependent, and required an ER stress response element (ERSE) and a nearby CCAAT box element. Electromobility shift assays and chromatin immunoprecipitation showed that ATF6 bound to the ERSE in the PDIA6 promoter, in vitro, and in the mouse heart, in vivo. Gain- and loss-of-function studies showed that PDIA6 protected cardiac myocytes against simulated ischemia/reperfusion-induced death in a manner that was dependent on the catalytic activity of PDIA6. Thus, by facilitating disulfide bond formation, and enhanced ER protein folding, PDIA6 may contribute to the protective effects of ATF6 in the ischemic mouse heart.

Regulation of microRNA expression in the heart by the ATF6 branch of the ER stress response.

A nodal regulator of endoplasmic reticulum stress is the transcription factor, ATF6, which is activated by ischemia and protects the heart from ischemic damage, in vivo. To explore mechanisms of ATF6-mediated protection in the heart, a whole-genome microRNA (miRNA) array analysis of RNA from the hearts of ATF6 transgenic (TG) mice was performed. The array identified 13 ATF6-regulated miRNAs, eight of which were downregulated, suggesting that they could contribute to increasing levels of their mRNAs. The down-regulated miRNAs, including miR-455, were predicted to target 45 mRNAs that we had previously shown by microarray analysis to be up-regulated by ATF6 in the heart. One of the miR-455 targets was calreticulin (Calr), which is up-regulated in the pathologic heart, where it modulates hypertrophic growth, potentially reducing the impact of the pathology. To validate the effects of miR-455, we showed that Calr protein was increased by ATF6 in mouse hearts, in vivo. In cultured cardiac myocytes, treatment with the ER stressor, tunicamycin, or with adenovirus encoding activated ATF6 decreased miR-455 and increased Calr levels, consistent with the effects of ATF6 on miR-455 and Calr, in vivo. Moreover, transfection of cultured cardiac myocytes with a synthetic precursor, premiR-455, decreased Calr levels, while transfection with an antisense, antimiR-455, increased Calr levels. The results of this study suggest that ER stress can regulate gene expression via ATF6-mediated changes in micro-RNA levels. Moreover, these findings support the hypothesis that ATF6-mediated down-regulation of miR-455 augments Calr expression, which may contribute to the protective effects of ATF6 in the heart.

Roles for endoplasmic reticulum-associated degradation and the novel endoplasmic reticulum stress response gene Derlin-3 in the ischemic heart.

RATIONALE: Stresses, such as ischemia, impair folding of nascent proteins in the rough endoplasmic reticulum (ER), activating the unfolded protein response, which restores efficient ER protein folding, thus leading to protection from stress. In part, the unfolded protein response alleviates ER stress and cell death by increasing the degradation of terminally misfolded ER proteins via ER-associated degradation (ERAD). ERAD is increased by the ER stress modulator, activating transcription factor (ATF)6, which can induce genes that encode components of the ERAD machinery. OBJECTIVE: Recently, it was shown that the mouse heart is protected from ischemic damage by ATF6; however, ERAD has not been studied in the cardiac context. A recent microarray study showed that the Derlin-3 (Derl3) gene, which encodes an important component of the ERAD machinery, is robustly induced by ATF6 in the mouse heart. METHODS AND RESULTS: In the present study, activated ATF6 induced Derl3 in cultured cardiomyocytes, and in the heart, in vivo. Simulated ischemia (sI), which activates ER stress, induced Derl3 in cultured myocytes, and in an in vivo mouse model of myocardial infarction, Derl3 was also induced. Derl3 overexpression enhanced ERAD and protected cardiomyocytes from simulated ischemia-induced cell death, whereas dominant-negative Derl3 decreased ERAD and increased simulated ischemia-induced cardiomyocyte death. CONCLUSIONS: This study describes a potentially protective role for Derl3 in the heart, and is the first to investigate the functional consequences of enhancing ERAD in the cardiac context.

Ischemia activates the ATF6 branch of the endoplasmic reticulum stress response.

Stresses that perturb the folding of nascent endoplasmic reticulum (ER) proteins activate the ER stress response. Upon ER stress, ER-associated ATF6 is cleaved; the resulting active cytosolic fragment of ATF6 translocates to the nucleus, binds to ER stress response elements (ERSEs), and induces genes, including the ER-targeted chaperone, GRP78. Recent studies showed that nutrient and oxygen starvation during tissue ischemia induce certain ER stress response genes, including GRP78; however, the role of ATF6 in mediating this induction has not been examined. In the current study, simulating ischemia (sI) in a primary cardiac myocyte model system caused a reduction in the level of ER-associated ATF6 with a coordinate increase of ATF6 in nuclear fractions. An ERSE in the GRP78 gene not previously shown to be required for induction by other ER stresses was found to bind ATF6 and to be critical for maximal ischemia-mediated GRP78 promoter induction. Activation of ATF6 and the GRP78 promoter, as well as grp78 mRNA accumulation during sI, were reversed upon simulated reperfusion (sI/R). Moreover, dominant-negative ATF6, or ATF6-targeted miRNA blocked sI-mediated grp78 induction, and the latter increased cardiac myocyte death upon simulated reperfusion, demonstrating critical roles for endogenous ATF6 in ischemia-mediated ER stress activation and cell survival. This is the first study to show that ATF6 is activated by ischemia but inactivated upon reperfusion, suggesting that it may play a role in the induction of ER stress response genes during ischemia that could have a preconditioning effect on cell survival during reperfusion.

Mesencephalic astrocyte-derived neurotrophic factor is an ischemia-inducible secreted endoplasmic reticulum stress response protein in the heart.

The endoplasmic reticulum (ER) stress response (ERSR) is activated when folding of nascent proteins in the ER lumen is impeded. Myocardial ischemia was recently shown to activate the ERSR; however, the role of this complex signaling system in the heart is not well understood. ER stress activates the transcription factor ATF6, which induces expression of proteins targeted to the ER, where they restore protein folding, thus fostering cytoprotection. We previously developed a transgenic mouse line that expresses a conditionally activated form of ATF6 in the heart. In this mouse line, ATF6 activation decreased ischemic damage in an ex vivo model of myocardial ischemia/reperfusion and induced numerous genes, including mesencephalic astrocyte-derived neurotrophic factor (MANF). In the present study, MANF expression was shown to be induced in cardiac myocytes and in other cell types in the hearts of mice subjected to in vivo myocardial infarction. Additionally, simulated ischemia induced MANF in an ATF6-dependent manner in neonatal rat ventricular myocyte cultures. In contrast to many other ER-resident ERSR proteins, MANF lacks a canonical ER-retention sequence, consistent with our finding that MANF was readily secreted from cultured cardiac myocytes. Knockdown of endogenous MANF with micro-RNA increased cell death upon simulated ischemia/reperfusion, whereas addition of recombinant MANF to media protected cultured cardiac myocytes from simulated ischemia/reperfusion-mediated death. Thus, a possible function of the ERSR in the heart is the ischemia-mediated induction of secreted proteins, such as MANF, that can function in an autocrine/paracrine manner to modulate myocardial damage from ER stresses, including ischemia.

Coordination of growth and endoplasmic reticulum stress signaling by regulator of calcineurin 1 (RCAN1), a novel ATF6-inducible gene.

Exposing cells to conditions that modulate growth can impair endoplasmic reticulum (ER) protein folding, leading to ER stress and activation of the transcription factor, ATF6. ATF6 binds to ER stress response elements in target genes, inducing expression of proteins that enhance the ER protein folding capacity, which helps overcome the stress and foster survival. To examine the mechanism of ATF6-mediated survival in vivo, we developed a transgenic mouse model that expresses a novel conditionally activated form of ATF6. We previously showed that activating ATF6 protected the hearts of ATF6 transgenic mice from ER stresses. In the present study, transcript profiling identified modulatory calcineurin interacting protein-1 (MCIP1), also known as regulator of calcineurin 1 (RCAN1), as a novel ATF6-inducible gene that encodes a known regulator of calcineurin/nuclear factor of activated T cells (NFAT)-mediated growth and development in many tissues. The ability of ATF6 to induce RCAN1 in vivo was replicated in cultured cardiac myocytes, where adenoviral (AdV)-mediated overexpression of activated ATF6 induced the RCAN1 promoter, up-regulated RCAN1 mRNA, inhibited calcineurin phosphatase activity, and exerted a striking growth modulating effect that was inhibited by RCAN1-targeted small interfering RNA. These results demonstrate that RCAN1 is a novel ATF6 target gene that may coordinate growth and ER stress signaling pathways. By modulating growth, RCAN1 may reduce the need for ER protein folding, thus helping to overcome the stress and enhance survival. Moreover, these results suggest that RCAN1 may also be a novel integrator of growth and ER stress signaling in many other tissues that depend on calcineurin/NFAT signaling for optimal growth and development.

Effects of the isoform-specific characteristics of ATF6 alpha and ATF6 beta on endoplasmic reticulum stress response gene expression and cell viability.

The endoplasmic reticulum (ER)-transmembrane proteins, ATF6 alpha and ATF6 beta, are cleaved during the ER stress response (ERSR). The resulting N-terminal fragments (N-ATF6 alpha and N-ATF6 beta) have conserved DNA-binding domains and divergent transcriptional activation domains. N-ATF6 alpha and N-ATF6 beta translocate to the nucleus, bind to specific regulatory elements, and influence expression of ERSR genes, such as glucose-regulated protein 78 (GRP78), that contribute to resolving the ERSR, thus, enhancing cell viability. We previously showed that N-ATF6 alpha is a rapidly degraded, strong transcriptional activator, whereas beta is a slowly degraded, weak activator. In this study we explored the molecular basis and functional impact of these isoform-specific characteristics in HeLa cells. Mutants in the transcriptional activation domain or DNA-binding domain of N-ATF6 alpha exhibited loss of function and increased expression, the latter of which suggested decreased rates of degradation. Fusing N-ATF6 alpha to the mutant estrogen receptor generated N-ATF6 alpha-MER, which, without tamoxifen exhibited loss-of-function and high expression, but in the presence of tamoxifen N-ATF6 alpha-MER exhibited gain-of-function and low expression. N-ATF6 beta conferred loss-of-function and high expression to N-ATF6 alpha, suggesting that ATF6 beta is an endogenous inhibitor of ATF6 alpha. In vitro DNA binding experiments showed that recombinant N-ATF6 beta inhibited the binding of recombinant N-ATF6 alpha to an ERSR element from the GRP78 promoter. Moreover, siRNA-mediated knock-down of endogenous ATF6 beta increased GRP78 promoter activity and GRP78 gene expression, as well as augmenting cell viability. Thus, the relative levels of ATF6 alpha and -beta, may contribute to regulating the strength and duration of ATF6-dependent ERSR gene induction and cell viability.

Alterations in oxidative phosphorylation complex proteins in the hearts of transgenic mice that overexpress the p38 MAP kinase activator, MAP kinase kinase 6.

Ischemia-reperfusion (I/R) has critical consequences in the heart. Recent studies on the functions of I/R-activated kinases, such as p38 mitogen-activated protein kinase (MAPK), showed that I/R injury is reduced in the hearts of transgenic mice that overexpress the p38 MAPK activator MAPK kinase 6 (MKK6). This protection may be fostered by changes in the levels of many proteins not currently known to be regulated by p38. To examine this possibility, we employed the multidimensional protein identification technology MudPIT to characterize changes in levels of proteins in MKK6 transgenic mouse hearts, focusing on proteins in mitochondria, which play key roles in mediating I/R injury in the heart. Of the 386 mitochondrial proteins identified, the levels of 58 were decreased, while only 2 were increased in the MKK6 transgenic mouse hearts. Among those that were decreased were 21 mitochondrial oxidative phosphorylation complex proteins, which was unexpected because p38 is not known to mediate such decreases. Immunoblotting verified that proteins in each of the five oxidative phosphorylation complexes were reduced in MKK6 mouse hearts. On assessing functional consequences of these reductions, we found that MKK6 mouse heart mitochondria exhibited 50% lower oxidative respiration and I/R-mediated reactive oxygen species (ROS) generation, both of which are predicted consequences of decreased oxidative phosphorylation complex proteins. Thus the cardioprotection observed in MKK6 transgenic mouse hearts may be partly due to decreased electron transport, which is potentially beneficial, because damaging ROS are known to be generated by mitochondrial complexes I and III during reoxygenation.