Scaffolding Protein Connector Enhancer of Kinase Suppressor of Ras 1 (CNKSR1) Regulates MAPK Inhibition Responsiveness in Pancreas Cancer via Crosstalk with AKT Signaling.

Combinatorial molecular therapy in pancreatic ductal adenocarcinoma (PDAC) has yielded largely disappointing results in clinical testing to-date as a multitude of adaptive resistance mechanisms is making selection of patients via molecular markers that capture essential, intersecting signaling routes challenging. Here, we report the scaffolding protein connector enhancer of kinase suppressor of Ras 1 (CNKSR1) as mediator of resistance to MAPK (MEK) inhibition. MEK inhibition in CNKSR1high cancer cells induces translocation of CNKSR1 to the plasma membrane where the scaffolding protein interacts with and stabilizes the phosphorylated form of AKT. CNKSR1-mediated AKT activation following MEK inhibition was associated with increased cellular p-PRAS40 levels and reduced nuclear translocation and cellular levels of FoxO1, a negative regulator of AKT signaling. In clinical PDAC specimens, high cytoplasmatic CNKSR1 levels correlated with increased cellular phospho-AKT and mTOR levels. Pharmacological co-blockade of AKT and MEK ranked top in induced synergies with MEK inhibition in CNKSR1high pancreas cancer cells among other inhibitor combinations targeting known CNKSR1 signaling. In vivo, CNKSR1high pancreatic tumors treated with AKT and MEK inhibitors showed improved outcome in the combination arm compared with single-agent treatment, an effect not observed in CNKSR1low models.Our results identify CNKSR1 as regulator of adaptive resistance to MEK inhibition by promoting crosstalk to AKT signaling via a scaffolding function for the phosphorylated form of AKT. CNSKR1 expression might be a possible molecular marker to enrich patients for future AKT-MEK inhibitor precision medicine studies. IMPLICATIONS: The CNKSR1 scaffold, identified within an RNAi screen as a novel mediator of resistance to MEK inhibition in pancreas cancer, connects the MAPK pathway and AKT signaling and may be adopted as a biomarker to select patients for combined MEK AKT blockade.

Atypical KRASG12R Mutant Is Impaired in PI3K Signaling and Macropinocytosis in Pancreatic Cancer.

Allele-specific signaling by different KRAS alleles remains poorly understood. The KRAS G12R mutation displays uneven prevalence among cancers that harbor the highest occurrence of KRAS mutations: It is rare (∼1%) in lung and colorectal cancers, yet relatively common (∼20%) in pancreatic ductal adenocarcinoma (PDAC), suggesting context-specific properties. We evaluated whether KRASG12R is functionally distinct from the more common KRASG12D- or KRASG12V-mutant proteins (KRASG12D/V). We found that KRASG12D/V but not KRASG12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRASG12D/V- but not KRASG12R-mutant PDAC. Surprisingly, we found that KRASG12R is defective for interaction with a key effector, p110α PI3K (PI3Kα), due to structural perturbations in switch II. Instead, upregulated KRAS-independent PI3Kγ activity was able to support macropinocytosis in KRASG12R-mutant PDAC. Finally, we determined that KRASG12R-mutant PDAC displayed a distinct drug sensitivity profile compared with KRASG12D-mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy. SIGNIFICANCE: We determined that KRASG12R is impaired in activating a key effector, p110α PI3K. As such, KRASG12R is impaired in driving macropinocytosis. However, overexpression of PI3Kγ in PDAC compensates for this deficiency, providing one basis for the prevalence of this otherwise rare KRAS mutant in pancreatic cancer but not other cancers.See related commentary by Falcomatà et al., p. 23.This article is highlighted in the In This Issue feature, p. 1.

A novel chimeric antigen receptor against prostate stem cell antigen mediates tumor destruction in a humanized mouse model of pancreatic cancer.

Despite advances in the understanding of its molecular pathophysiology, pancreatic cancer remains largely incurable, highlighting the need for novel therapies. We developed a chimeric antigen receptor (CAR) specific for prostate stem cell antigen (PSCA), a glycoprotein that is overexpressed in pancreatic cancer starting at early stages of malignant transformation. To optimize the CAR design, we used antigen-recognition domains derived from mouse or human antibodies, and intracellular signaling domains containing one or two T cell costimulatory elements, in addition to CD3zeta. Comparing multiple constructs established that the CAR based on human monoclonal antibody Ha1-4.117 had the greatest reactivity in vitro. To further analyze this CAR, we developed a human pancreatic cancer xenograft model and adoptively transferred CAR-engineered T cells into animals with established tumors. CAR-engineered human lymphocytes induced significant antitumor activity, and unlike what has been described for other CARs, a second-generation CAR (containing CD28 cosignaling domain) induced a more potent antitumor effect than a third-generation CAR (containing CD28 and 41BB cosignaling domains). While our results provide evidence to support PSCA as a target antigen for CAR-based immunotherapy of pancreatic cancer, the expression of PSCA on selected normal tissues could be a source of limiting toxicity.

A novel 3D fibril force assay implicates src in tumor cell force generation in collagen networks.

New insight into the biomechanics of cancer cell motility in 3D extracellular matrix (ECM) environments would significantly enhance our understanding of aggressive cancers and help identify new targets for intervention. While several methods for measuring the forces involved in cell-matrix interactions have been developed, previous to this study none have been able to measure forces in a fibrillar environment. We have developed a novel assay for simultaneously measuring cell mechanotransduction and motility in 3D fibrillar environments. The assay consists of a controlled-density fibrillar collagen gel atop a controlled-stiffness polyacrylamide (PAA) surface. Forces generated by living cells and their migration in the 3D collagen gel were measured with the 3D motion of tracer beads within the PAA layer. Here, this 3D fibril force assay is used to study the role of the invasion-associated protein kinase Src in mechanotransduction and motility. Src expression and activation are linked with proliferation, invasion, and metastasis, and have been shown to be required in 2D for invadopodia membranes to direct and mediate invasion. Breast cancer cell line MDA-MD-231 was stably transfected with GFP-tagged constitutively active Src or wild-type Src. In 3D fibrillar collagen matrices we found that, relative to wild-type Src, constitutively active Src: 1) increased the strength of cell-induced forces on the ECM, 2) did not significantly change migration speed, and 3) increased both the duration and the length, but not the number, of long membrane protrusions. Taken together, these results support the hypothesis that Src controls invasion by controlling the ability of the cell to form long lasting cellular protrusions to enable penetration through tissue barriers, in addition to its role in promoting invadopodia matrix-degrading activity.

Association of Over-Expressed Estrogen Receptor Alpha with Development of Tamoxifen Resistant Hyperplasia and Adenocarcinomas in Genetically Engineered Mice.

BACKGROUND: Estrogen receptor alpha (ERα) and cyclin D1 are frequently co-expressed in human breast cancer. Some, but not all, studies link tamoxifen resistance to co-expression of cyclin D1 and ERα. In mice over-expression of either cyclin D1 or ERα in mammary epithelial cells is sufficient to induce mammary hyperplasia. Cyclin D1 over-expression in mice leads to mammary adenocarcinoma associated with activated estrogen signaling pathways. ERα over-expression in mice leads to mammary hyperplasia and cancer. Significantly, disease development in these mice is abrogated by loss of cyclin D1. METHODS: Genetically engineered mouse models were used to determine whether or not ERα over-expression demonstrated cooperativity with cyclin D1 over-expression in cancer development, reaction to the chemical carcinogen DMBA, or tamoxifen response. RESULTS: Adding ERα over-expression to cyclin D1 over-expression increased the prevalence of hyperplasia but not cancer. Single dose DMBA exposure did not increase cancer prevalence in any of the genotypes although cyclin D1 over-expressing mice demonstrated a significant increase in hyperplasia. Tamoxifen treatment was initiated at both young and older ages to test for genotype-specific differences in response. Although normal ductal structures regressed in all genotypes at both younger and older ages, tamoxifen did not significantly reduce the prevalence of either hyperplasia or cancer in any of the genotypes. All of the cancers that developed were hormone receptor positive, including those that developed on tamoxifen, and all showed expression of nuclear-localized cyclin D1. In summary, development of tamoxifen resistant hyperplasia and cancer was associated with expression of ERα and cyclin D1. CONCLUSION: These preclinical models will be useful to test strategies for overcoming tamoxifen resistance, perhaps by simultaneously targeting cell cycle regulatory pathways associated with cyclin D1.

Altered AIB1 or AIB1Δ3 expression impacts ERα effects on mammary gland stromal and epithelial content.

Amplified in breast cancer 1 (AIB1) (also known as steroid receptor coactivator-3) is a nuclear receptor coactivator enhancing estrogen receptor (ER)α and progesterone receptor (PR)-dependent transcription in breast cancer. The splice variant AIB1Δ3 demonstrates increased ability to promote ERα and PR-dependent transcription. Both are implicated in breast cancer risk and antihormone resistance. Conditional transgenic mice tested the in vivo impact of AIB1Δ3 overexpression compared with AIB1 on histological features of increased breast cancer risk and growth response to estrogen and progesterone in the mammary gland. Combining expression of either AIB1 or AIB1Δ3 with ERα overexpression, we investigated in vivo cooperativity. AIB1 and AIB1Δ3 overexpression equivalently increased the prevalence of hyperplastic alveolar nodules but not ductal hyperplasia or collagen content. When AIB1 or AIB1Δ3 overexpression was combined with ERα, both stromal collagen content and ductal hyperplasia prevalence were significantly increased and adenocarcinomas appeared. Overexpression of AIB1Δ3, especially combined with overexpressed ERα, led to an abnormal response to estrogen and progesterone with significant increases in stromal collagen content and development of a multilayered mammary epithelium. AIB1Δ3 overexpression was associated with a significant increase in PR expression and PR downstream signaling genes. AIB1 overexpression produced less marked growth abnormalities and no significant change in PR expression. In summary, AIB1Δ3 overexpression was more potent than AIB1 overexpression in increasing stromal collagen content, inducing abnormal mammary epithelial growth, altering PR expression levels, and mediating the response to estrogen and progesterone. Combining ERα overexpression with either AIB1 or AIB1Δ3 overexpression augmented abnormal growth responses in both epithelial and stromal compartments.

Role of ERalpha in the differential response of Stat5a loss in susceptibility to mammary preneoplasia and DMBA-induced carcinogenesis.

Deregulated estrogen signaling is evidently linked to breast cancer pathophysiology, although the role of signal transducer and activator of transcription (Stat)5a, integral to normal mammary gland development, is less clear. A mouse model of mammary epithelial cell-targeted deregulated estrogen receptor alpha (ERalpha) expression [conditional ERalpha in mammary epithelium (CERM)] was crossed with mice carrying a germ line deletion of Stat5a [Stat5a-/-] to investigate interactions between ERalpha and Stat5a in mammary tissue. CERM, CERM/Stat5a+/-, CERM/Stat5a-/-, Stat5a+/-, Stat5a-/- and wild-type (WT) mice were generated to test the roles of ERalpha and Stat5a on pubertal differentiation and cancer progression with and without exposure to the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Only CERM/Stat5a-/- mice demonstrated delayed pubertal terminal end bud differentiation. Without DMBA exposure, Stat5a loss abrogated ERalpha-initiated hyperplastic alveolar nodule (HAN) development and, similarly, Stat5a-/- mice did not develop HANs. However, although Stat5a loss still reduced ERalpha-initiated HAN prevalence following DMBA exposure, Stat5a loss without deregulated ERalpha was associated with an increased HAN prevalence compared with WT. Progression to ERalpha(+) and ERalpha(-) adenocarcinoma was found in all CERM-containing genotypes (CERM, CERM/Stat5a+/-, CERM/Stat5a-/-) and ERalpha(+) adenocarcinoma in the Stat5a-/- genotype. The mammary epithelial cell proliferative index was increased only in CERM mice independent of Stat5a loss. No differences in apoptotic indices were found. In summary, Stat5a cooperated with deregulated ERalpha in retarding pubertal mammary differentiation and contributed to ERalpha-initiated preneoplasia, but its loss did not prevent development of invasive cancer. Moreover, in the absence of deregulated ERalpha, Stat5a loss was associated with development of both HANs and invasive cancer following DMBA exposure.

Simultaneous suppression of epidermal growth factor receptor and c-erbB-2 reverses aneuploidy and malignant phenotype of a human ovarian carcinoma cell line.

Coexpression of epidermal growth factor receptor (EGFR) and c-erbB-2 in 47-68% of ovarian cancer cells indicate their strong association with tumor formation. We examined the effects of simultaneous antisense- or immunosuppression of EGFR and c-erbB-2 expression on the invasive phenotype, aneuploidy, and genotype of cultured human ovarian carcinoma cells (NIH:OVCAR-8). We report here that suppression of both EGFR and c-erbB-2 results in regression of aneuploidy and genomic imbalances in NIH:OVCAR-8 cells, restores a more normal phenotype, and results in a more normal gene expression profile. Combined with cytogenetic analysis, our data demonstrate that the regression of aneuploidy is due to the selective apoptosis of double antisense transfected cells with highly abnormal karyotype.