Recent research has started to focus on identifying individuals who are at clinical high risk of developing psychosis as a means to try and understand the predictors and mechanisms involved in the progress to a full psychotic episode. The aim of the current study was to provide an initial description and prevalence rates of specific content found within attenuated positive symptoms. The Content of Attenuated Positive Symptoms (CAPS) codebook was used by independent raters to determine the presence of content within a sample of written vignettes. Krippendorff's alpha was used to determine inter-rater reliability. Overall, the majority of items fell in or above an acceptable range of reliability. There was heterogeneity present in the types of content endorsed. However, the most commonly endorsed items included being perplexed by reality, increased hypervigilence, being gifted, hearing indistinct and distinct sounds, seeing figures or shadows, something touching the individual, and unpleasant smells. The use of the CAPS codebook is a reliable way to code the content of attenuated positive symptoms. Identifying and monitoring the presence of certain content may provide insight into the presence of other comorbid issues and the potential for future conversion.

Drug resistance is a major barrier to successful cancer treatment. For patients with HER2-positive breast cancer who initially respond to therapy, the majority develop resistance within one year of treatment. Patient outcomes could improve significantly if we can find and exploit common mechanisms of acquired resistance to different targeted therapies. Overexpression of t-Darpp, a truncated form of the dual kinase/phosphatase inhibitor Darpp-32, has been linked to acquired resistance to trastuzumab, a front-line therapy for HER2-positive breast cancer. Darpp-32 reverses t-Darpp's effect on trastuzumab resistance. In this study, we examined whether t-Darpp could be involved in resistance to lapatinib, another HER2-targeted therapeutic. Lapatinib-resistant SKBR3 cells (SK/LapR) showed a marked change in the Darpp-32:t-Darpp ratio toward a predominance of t-Darpp. Overexpression of t-Darpp alone was not sufficient to confer lapatinib resistance, but cells that overexpress t-Darpp partially mimicked the molecular resistance phenotype observed in SK/LapR cells exposed to lapatinib. SK/LapR cells failed to down-regulate Survivin and failed to induce BIM accumulation in response to lapatinib; cells overexpressing t-Darpp exhibited only the failed BIM accumulation. t-Darpp knock-down reversed this phenotype. Using a fluorescence-based co-culture system, we found that cells overexpressing t-Darpp formed colonies in lapatinib within 3–4 weeks, whereas parental cells in the same co-culture did not. Overall, t-Darpp appears to mediate a survival advantage in lapatinib, possibly linked to failed lapatinib-induced BIM accumulation. t-Darpp might also be relevant to acquired resistance to other cancer drugs that rely on BIM accumulation to induce apoptosis.

t‐Darpp (truncated isoform of dopamine‐ and cAMP ‐regulated phosphoprotein) is a protein encoded by the PPP 1R1B gene and is expressed in breast, colon, esophageal, gastric, and prostate cancers, as well as in normal adult brain striatal cells. Overexpression of t‐Darpp in cultured cells leads to increased protein kinase A activity and increased phosphorylation of AKT (protein kinase B). In HER 2+ breast cancer cells, t‐Darpp confers resistance to the chemotherapeutic agent trastuzumab. To shed light on t‐Darpp function, we studied its secondary structure, oligomerization status, metal‐binding properties, and phosphorylation by cyclin‐dependent kinases 1 and 5. t‐Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn, and 35% random coil structures. It binds calcium, but not other metals commonly found in biological systems. The T39 site, critical for t‐Darpp activation of the AKT signaling pathway, is a substrate for phosphorylation by cyclin‐dependent kinase 1 and cyclin‐dependent kinase 5. Gel filtration chromatography, sedimentation equilibrium analysis, blue native gel electrophoresis, and glutaraldehyde‐mediated cross‐linking experiments demonstrate that the majority of t‐Darpp exists as a monomer, but forms low levels (< 3%) of hetero‐oligomers with its longer isoform Darpp‐32. t‐Darpp has a large Stokes radius of 4.4 nm relative to its mass of 19 kDa, indicating that it has an elongated structure.

t-Darpp is the truncated form of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (Darpp-32) and has been demonstrated to confer resistance to trastuzumab, a Her2-targeted anticancer agent, via sustained signaling through the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt pathway and activation of protein kinase A (PKA). The mechanism of t-Darpp-mediated PKA activation is poorly understood. In the PKA holoenzyme, when the catalytic subunits are bound to regulatory subunits RI or RII, kinase activity is inhibited. We investigated PKA activity and holoenzyme composition in cell lines overexpressing t-Darpp (SK.tDp) or a T39A phosphorylation mutant (SK.tDpT39A), as well as an empty vector control cell line (SK.empty). We also evaluated protein-protein interactions between t-Darpp and PKA catalytic (PKAc) or regulatory subunits RI and RII in those cell lines. SK.tDp cells had elevated PKA activity and showed diminished association of RI with PKAc, whereas SK.tDpT39A cells did not have these properties. Moreover, wild type t-Darpp associates with RI. Concurrent expression of Darpp-32 reversed t-Darrp’s effects on PKA holoenzyme state, consistent with earlier observations that Darpp-32 reverses t-Darpp’s activation of PKA. Together, t-Darpp phosphorylation at T39 seems to be crucial for t-Darpp—mediated PKA activation and this activation appears to occur through an association with RI and sequestering of RI away from PKAc. The t-Darpp-RI interaction could be a druggable target to reduce PKA activity in drug-resistant cancer.

Trastuzumab has led to improved survival rates of HER2+ breast cancer patients. However, acquired resistance remains a problem in the majority of cases. t-Darpp is over-expressed in trastuzumab-resistant cell lines and its over-expression is sufficient for conferring the resistance phenotype. Although its mechanism of action is unknown, t-Darpp has been shown to increase cellular proliferation and inhibit apoptosis. We have reported that trastuzumab-resistant BT.HerR cells that over-express endogenous t-Darpp are sensitized to EGFR inhibition in the presence (but not the absence) of trastuzumab. The purpose of the current study was to determine if t-Darpp might modulate sensitivity to EGFR inhibitors in trastuzumab-resistant cells. Using EGFR tyrosine kinase inhibitors AG1478, gefitinib and erlotinib, we found that trastuzumab-resistant SK.HerR cells were sensitized to EGFR inhibition, compared to SK-Br-3 controls, even in the absence of trastuzumab. t-Darpp knock-down in SK.HerR cells reversed their sensitivity to EGFR inhibition. Increased EGFR sensitivity was also noted in SK.tDp cells that stably over-express t-Darpp. High levels of synergy between trastuzumab and the EGFR inhibitors were observed in all cell lines with high t-Darpp expression. These cells also demonstrated more robust activation of EGFR signaling and showed greater EGFR stability than parental cells. The T75A phosphorylation mutant of t-Darpp did not confer sensitivity to EGFR inhibition nor activation of EGFR signaling. The over-expression of t-Darpp might facilitate enhanced EGFR signaling as part of the trastuzumab resistance phenotype. This study suggests that the presence of t-Darpp in HER2+ cancers might predict the enhanced response to dual HER2/EGFR targeting.

Cell-based therapies have intriguing potential for the treatment of a variety of neurological disorders. One such example is genetically engineered cytotoxic T lymphocytes (CTLs) that are being investigated in brain tumor clinical trials. The development of methods for CTL delivery is critical to their use in the laboratory and clinical setting. In our study, we determined whether CTLs can migrate through fibrin matrices and if their migration, survival, and function could be modulated by adding chemokines to the matrix. Our results indicated that CTLs can freely migrate through fibrin matrices. As expected, the addition of the monocyte chemotactic protein-1 (MCP-1), also known as chemokine C-C motif ligand 2 (CCL2), to the surrounding media increased egress of the CTLs out of the fibrin clot. Interleukin (IL) -2 and/or IL-15 embedded in the matrix enhanced T cell survival and further promoted T cell migration. The interleukin-13 receptor alpha 2 specific (IL-13R alpha2) T cells that traveled out of the fibrin clot retained the capacity to kill U251 glioma cells. In summary, CTLs can survive and migrate robustly in fibrin matrices. These processes can be influenced by modification of matrix constituents. We conclude that fibrin matrices may be suitable T cell carriers and can be used to facilitate understanding of T cell interaction with the surrounding microenvironment.

t-Darpp is a protein encoded by the PPP1R1B gene and is expressed in breast, colon, esophageal, gastric, and prostate cancers, as well as in normal adult brain striatal cells.Overexpression of t-Darpp in cultured cells leads to increased protein kinase A activity and increased phosphorylation of AKT (protein kinase B). In HER2+ breast cancer cells t-Darpp confers resistance to the chemotherapeutic agent trastuzumab. To shed light on t-Darpp function, we studied its secondary structure, oligomerization status, metal-binding properties, and phosphorylation by cyclin dependent kinases 1 and 5. t-Darpp exhibits 12% alpha helix, 29% beta strand, 24% beta turn and 35% random coil structures. t-Darpp binds to calcium, but not to other metals commonly found in biological systems. The T39 site, critical for t-Darpp activation of the AKT signaling pathway, is a substrate for phosphorylation by cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 5 (CDK5). Gel filtration chromatography, sedimentation equilibrium analysis, blue native gel electrophoresis, and glutaraldehyde-mediated crosslinking experiments demonstrate that the majority of t-Darpp exists as a monomer, but forms low levels (< 3%) of hetero-oligomers with its longer isoform Darpp-32. t-Darpp has a large Stokes radius of 4.4 nm relative to its mass of 19 kDa, indicating that it has an elongated structure.

Background: We have previously shown that levels of the 32kDa dopamine and cAMP-regulated phosphoprotein Darpp-32 (Dp32) and its amino-truncated isoform t-Darpp (tDp) are altered in trastuzumab-resistant HER2+ breast cancer cells. Specifically, tDp is upregulated in models of trastuzumab resistance and over-expression of the protein is sufficient to confer trastuzumab resistance. Dp32 antagonizes the effect of tDp, exhibiting overall growth inhibitory properties in breast cancer cell lines. In neuronal cells, Dp32 is regulated by phosphorylation at two key threonine residues, T34 and T75. The T34 site is present only in Dp23 as tDp is truncated by the first 39 amino acids. The latter site is phosphorylated predominantly by cdk5 in vivo and by either cdk5 or cdk1 in vitro. It is not known whether tDp is similarly phosphorylated or if the same kinases act in breast cancer cells as in neuronal cells. Because we have preliminary data suggesting an interaction between tDp and cdk1 (but not cdk5) in HER2+ breast cancer cells, we investigated whether cdk1 or cdk5 could phosphorylate tDp at the T75 site. Methods: We performed in vitro kinase assays using recombinant tDp and commercial cdk1 and cdk5 enzymes. Phosphorylation was also examined in cells that overexpress endogenous tDp (SK/HerR and BT/HerR cells), that overexpress exogenous tDp (SK.tDp cells) or that express exogenous tDp and Dp32 (SK.dDp). Cells were treated with either RO-3306, a cdk1 inhibitor, or Roscovitine, a pan-cdk inhibitor with preference for cdk5, and assayed by Western blotting for T75 phosphorylation. All experiments were repeated three times and calculated for statistical significance. Results: In vitro kinase assays demonstrated that both cdk1 and cdk5 were capable of phosphorylating tDp at the T75 residue, with an apparent preferential activity by cdk1. In cell lines, RO-3306 led to decreased T75 phosphorylation within one hour. The effect of RO-3306 was also confirmed in a dose-dependent manner with optimal inhibition at a concentration of 10 μM. Roscovitine also led to decreased T75 phosphorylation at a concentration of 20 μM. In cells that express both Dp32 and tDp, the cdk inhibitors led to decreased phosphorylation of both proteins to similar extents. Conclusion: Our study provides new insight into cdk1-mediated regulation of tDp and Dp32. We demonstrate that both cdk1 and cdk5 are able to phosphorylate these proteins but that cdk1 appears to be the predominant mediator of phosphorylation in breast cancer cell lines. A possible competition might occur between Dp32 and tDp, given their similar propensity for phosphorylation by cdk1. Further examination of the relationship between the two kinases and their activities toward tDp and Dp32 will help us delineate the mechanism by which tDp mediates trastuzumab resistance and Dp32 antagonizes this phenotype. Citation Format: Erin Denny, Arianna Celis, Jamil Momand, Susan E. Kane. t-Darpp is phosphorylated by cdk1 and cdk5 in HER2+ breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A058.