Darpp-32 and Its Truncated Variant t-Darpp Have Antagonistic Effects on Breast Cancer Cell Growth and Herceptin Resistance

BACKGROUND: Herceptin (trastuzumab) is a humanized monoclonal antibody that is approved for the treatment of metastatic breast cancer patients whose tumors overexpress Her2 (erbB2/neu). Up to 70% of Her2-positive breast cancers demonstrate a response to Herceptin-based therapies, but resistance almost inevitably arises within a year of the initial response. To help understand the mechanism of Herceptin resistance, we isolated clonal variants of Her2-positive BT474 human breast cancer cells (BT/Her(R)) that are highly resistant to Herceptin. These cell lines exhibit sustained PI3K/Akt signaling as an essential component of Herceptin-resistant proliferation. Several genes in the protein kinase A (PKA) signaling network have altered expression in BT/Her(R) cells, including PPP1R1B, which encodes a 32 kDa protein known as Darpp-32 and its amino-terminal truncated variant, t-Darpp. The purpose of the current work was to determine the role of Darpp-32 and t-Darpp in Herceptin resistance. METHODOLOGY AND RESULTS: We determined expression of Darpp-32 and t-Darpp in BT/Her(R) cells selected for resistance to Herceptin. Subsequently, cDNAs encoding the two isoforms of Darpp-32 were transfected, separately and together, into Her2-positive SK-Br-3 breast cancer cells. Transfected cells were tested for resistance to Herceptin and Herceptin-mediated dephosphorylation of Akt. DNA binding activity by the cAMP response element binding protein (CREB) was also measured. We found that BT/Her(R) cells overexpressed t-Darpp but not Darpp-32. Moreover, t-Darpp overexpression in SK-Br-3 cells was sufficient for conferring resistance to Herceptin and Herceptin-mediated dephosphorylation of Akt. Darpp-32 co-expression reversed t-Darpp's effects on Herceptin resistance and Akt phosphorylation. t-Darpp overexpression led to increased CREB binding activity, which was also reversible by Darpp-32. CONCLUSIONS: t-Darpp and Darpp-32 appear to have antagonistic effects on Herceptin resistance. We present a unified model by which these effects might be mediated via the PKA regulatory network

Yellow nail syndrome with chylothorax after coronary artery bypass grafting

Abstract Background Yellow nail syndrome is a rare condition considered secondary to functional anomalies of lymphatic drainage. Yellow nail syndrome is diagnosed through the triad of intrathoracic findings (30% being pleural effusions), nail discoloration, and lymphedema, with any two features sufficient for diagnosis. We report the second case of post-operative yellow nail syndrome. Case presentation After coronary artery bypass grafting, our patient presented with chylothorax on post-operative day 13 and yellow toenail discoloration on post-operative day 28, diagnosing yellow nail syndrome. Initial conservative management with pigtail catheter drainage and low-fat diet with medium-chain triglycerides reduced chylous drainage from 350 mL/day on post-operative day 14 to < 100 mL/day on post-operative day 17. However, by post-operative day 18, drainage returned to 350 mL/day that persisted despite attempts to readjust the catheter position, replacement of catheter with chest tube, and transition to total parenteral nutrition and octreotide while nil per os. Lymphangiogram on post-operative day 32 did not identify the thoracic duct or cisterna chyli, precluding embolization. Talc and doxycycline pleurodeses performed on post-operative days 33 and 38, respectively, resolved his chylothorax and nail discoloration. Conclusions Both yellow nail syndrome and chylothorax as a complication of coronary artery bypass grafting are rare entities. The proposed mechanism of post-operative chylothorax is iatrogenic injury to thoracic duct or collateral lymphatic vessels. Diagnosing yellow nail syndrome in patients with post-operative chylothorax (through co-existing yellow nail discoloration and/or lymphedema) may suggest predisposition to impaired lymphatic drainage, portending a difficult recovery and potentially indicating need for surgical management

Expression of Darpp-32 and t-Darpp in BT/Her^R cells.

<p>(A) Expression of Darpp-32 (open bars) and t-Darpp (filled bars) mRNA in BT474 cells and BT/Her^R clones was analyzed by a SYBR Green assay. Shown are the average expression levels (±S.D.) of Darpp-32 and t-Darpp mRNAs in each indicated cell line relative to the corresponding mRNA levels in BT474 cells. Quadruplicate measurements were made on a single isolation of RNA from each cell line analyzed. Statistically significant differences from BT cells were determined by two-tailed t-test. *, p<0.05; **, p<0.01, ***, p<0.001. (B) Expression of Darpp-32 and t-Darpp proteins in BT474 and Her^R/BT clones was analyzed by Western hybridization. SK-Br-3 cells exogenously expressing both t-Darpp and Darpp-32 (tDp/Dp32) were used as control for the respective forms of the protein; this lane was loaded with one-fifth as much lysate as the other lanes to account for their high exogenous t-Darpp/Darpp-32 expression. The top panel shows a short-exposure image of the membrane stained with an antibody (Santa Cruz H-62) that recognizes both Darpp-32 and t-Darpp. The middle panel is a long exposure of the same membrane and the bottom image shows the levels of actin as a loading control. Numbers below the actin image indicate the ratio of Darpp-32 to t-Darpp signal for each lane in the middle panel, as determined by densitometric scanning. Clones shown in this figure and additional BT/Her^R clones have been analyzed multiple times by Western analysis, always with very high t-Darpp expression and low or undetectable expression of Darpp-32.</p

A working model of Herceptin resistance in BT/Her^R cells.

<p>Several intracellular changes, including down-regulation of PKA-RIIα, PKIγ and PTG (green arrows) and up-regulation of t-Darpp (red arrow) work coordinately to enhance PKA activity. PKA, in turn, either activates the PI3K/Akt pathway (possibly through EGFR or the p85 regulatory subunit of PI3K) or promotes sustained phospho-Akt levels by activating the PP-1 inhibitory activity of Darpp-32. This activity is stimulated via a phosphorylation event at Thr-34, which is absent from t-Darpp. A second phosphorylation event, at Thr-75, activates Darpp-32 as a PKA inhibitor. We speculate that t-Darpp may interfere with this PKA inhibition via a dominant negative mechanism. Dashed lines indicate activities that are down-regulated in BT/Her^R cells relative to BT474 cells. Question marks indicate hypothetical pathways that are activated in BT/Her^R cells. All other pathways represent well established activities associated with the indicated proteins and enzymes. Abbreviations not already cited: PDK1/2, 3-phosphoinositide-dependent kinase-1/2; Pten, phosphatase and tensin homologue deleted in chromosome 10; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate.</p

Overexpression of t-Darpp confers Herceptin resistance.

<p>(A) SK-Br-3 cells transfected with an empty pcDNA3.0/Neo vector (Neo1 and Neo2) or with a pcDNA3.0/t-Darpp vector (tDp.A, J, L, and O) were analyzed by Western hybridization using the anti-Darpp-32 antibody (H-62) to determine expression of t-Darpp in the transfected cells. (B) Growth of the indicated cell lines was analyzed in the presence or absence of 0.1 µM or 0.3 µM Herceptin, as indicated, for 7 days using a Sulforhodamine B (SRB) assay. Shown are the absorbances of the cells (average of quadruplicate platings, +S.D.) incubated in the presence of Herceptin, normalized to the same cells grown in the absence of Herceptin. (C) Western hybridization was used to measure total Akt and phospho-Akt levels in the indicated clones at various times after the addition of 0.2 µM Herceptin. This experiment was performed twice on the clones shown in this figure and once on an additional clone not shown. Similar results were obtained in both experiments, although clone tDp.A also showed suppression of phospho-Akt levels at the 1 hr time point in the other experiment, with full recovery by 2 hrs (see text).</p

t-Darpp and Darpp-32 have antagonistic effects on CRE binding activity.

<p>Nuclear CRE binding activity in parent SK-Br-3 cells (SK) and the indicated transfected clones was analyzed by EMSA, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006220#s2" target="_blank">Materials and Methods</a>. Quantification of band density, relative to the SK lane, is shown below each lane. The experiment was performed twice and the relative band densities for the other experiment were 1.9 for tDp.A and 1.1, 0.5 and 0.4 for the three tDp/Dp32 clones, respectively.</p

Preclinical curriculum of prospective case-based teaching with faculty- and student-blinded approach

Abstract Background Case-based teaching with real patient cases provides benefit of simulating real-world cognition. However, while clinical practice involves a prospective approach to cases, preclinical instruction typically involves full disclosure of case content to faculty, introducing hindsight bias into faculty teaching in medical curricula. Methods During 2015–2018, we piloted an optional medical school curriculum involving 6–7 one-hour sessions over a 3-month period each year. New groups enrolled each year from first- and second-year classes. A facilitator provided a blinded physician discussant and blinded students with case information during and not in advance of each session, allowing prospective case-based discussions. Cases were based on real patients treated in the Department of Medicine. Clinical material was presented in the chronologic sequence encountered by treating physicians. Content covered a median of 5 patient visits/case (range: 2–10) spanning over months. A 14-item survey addressing components of the reporter-interpreter-manager-educator (RIME) scheme was developed and used to compare self-reported clinical skills between course participants and non-participant controls during the 2016 course iteration. Results This elective curriculum at Stanford School of Medicine involved 170 preclinical students (22.7% of 750 eligible). During the 2016 course iteration, a quasi-experimental study compared self-reported clinical skills between 29 course participants (response rate: 29/49 [59.2%]) and 35 non-participant controls (response rate: 35/132 [26.5%]); students self-assessed clinical skills via the RIME-based survey developed for the course. Two-sample t-tests compared the change in pre- and post-course skills between course participants and non-participants. Of 15 Department of Medicine faculty members invited as discussants, 12 (80%) consented to participate. Compared with controls, first-year participants self-assessed significantly greater improvement in understanding how clinicians reason through cases step-by-step to arrive at diagnoses (P = 0.049), work through cases in longitudinal settings (P = 0.049), and share information with patients (P = 0.047). Compared with controls, second-year participants self-assessed significantly greater improvement (P = 0.040) in understanding how clinicians reason through cases step-by-step to arrive at diagnoses. Conclusions Prospective case-based discussions with blinding of faculty and students to clinical content circumvents hindsight bias and may impart real-world cognitive skills as determined by student self-report

Impact of modern chemotherapy on the survival of women presenting with <it>de novo</it> metastatic breast cancer

<p>Abstract</p> <p>Background</p> <p>Data that directly associate utilization of novel systemic therapies with survival trends in metastatic breast cancer (MBC) are limited. In the setting of <it>de novo</it> MBC, large registry analyses cite positive temporal trends in survival, but the extent to which advances in systemic therapy have contributed to these gains is not clear.</p> <p>Methods</p> <p>The City of Hope Cancer Registry was used to identify a consecutive series of patients with <it>de novo</it> MBC who received their first line of therapy between 1985 and 2004. Comprehensive clinicopathologic and treatment-related data were collected for each patient. Univariate analyses were conducted via Cox regression to identify factors associated with improved survival. Multivariate analysis was also conducted via Cox regression and the stepwise procedure was used to identify independent predictors of survival.</p> <p>Results</p> <p>A total of 324 patients with <it>de novo</it> MBC were identified. After application of exclusion criteria, including the sole presence of supraclavicular node metastasis, 274 patients were retained in the analysis. The treatment-related characteristics associated with improved survival included: use of endocrine therapy (hazard ratio [HR] 0.60, 95%CI 0.47-0.77; P<0.0001), and addition of bisphosphonates (HR 0.70, 95%CI 0.52-0.96; P=0.02). However, recipients of novel cytotoxic agents (defined as drugs approved for MBC since 1994) had no improvement in survival relative to patients treated with older cytotoxic agents. On multivariate analysis, age (< 50), receipt of aromatase inhibitors, and receipt of zoledronic acid were independent predictors of survival.</p> <p>Conclusions</p> <p>The overall survival of women with <it>de novo</it> metastatic breast cancer has improved over the past 20 years. However, the contribution of conventional cytotoxic agents to this improvement is minimal.</p