Citations

Abstract Prostate cancer is the most frequently diagnosed male cancer. For prostate cancer that has progressed to an advanced or metastatic stage, androgen deprivation therapy (ADT) is the standard of care. ADT inhibits activity of the androgen receptor (AR), a master regulator transcription factor in normal and cancerous prostate cells. The major limitation of ADT is the development of castration-resistant prostate cancer (CRPC), which is almost invariably due to transcriptional re-activation of the AR. One mechanism of AR transcriptional re-activation is expression of AR-V7, a truncated, constitutively active AR variant (AR-V) arising from alternative AR pre-mRNA splicing. Noteworthy, AR-V7 is being developed as a predictive biomarker of primary resistance to androgen receptor (AR)-targeted therapies in CRPC. Multiple additional AR-V species are expressed in clinical CRPC, but the extent to which these may be co-expressed with AR-V7 or predict resistance is not known. Here we utilized long read sequencing to identify and quantify AR isoforms expressed in CRPC. To unambiguously characterize all AR isoforms, we prepared Iso-Seq™ libraries via 3’ rapid amplification of cDNA ends (RACE) with RNA isolated from prostate cancer cell lines and xenograft tissues using a forward primer anchored in AR exon 1. 3’RACE reactions were subjected to single molecule, real-time (SMRT®) long-read sequencing with a Pacific Biosciences RSII System. Our work identified AR-V9 as a truncated isoform that is frequently co-expressed with AR-V7 in CRPC. Mechanistically, our work re-annotated AR-V7 and AR-V9 mRNAs, showing these two species shared a common 3’ terminal exon containing separate splice acceptor sites. Taking into account this new information, novel siRNAs and antibodies which could distinguish between AR-V7 and AR-V9 were designed, validated and used to measure the relative expression of these two AR isoforms in CRPC cells with a view to determining the potential of AR-V9 as a predictive biomarker of primary resistance to AR-targeted therapies. Citation Format: Manish Kohli, Yeung Ho, David W. Hillman, Jamie L. Van Etten, Christine Henzler, Rendong Yang, Yingming Li, Elizabeth Tseng, Ting Hon, Tyson A. Clark, Liguo Wang, Kevin Silverstein, Liewei Wang, Scott M. Dehm. SMRT® Sequencing of full-length androgen receptor isoforms in prostate cancer reveals previously hidden drug resistant variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 425. doi:10.1158/1538-7445.AM2017-425

Abstract LIN28 is well known as a RNA-binding protein and a suppressor of microRNA biogenesis, by selectively blocking the processing of let-7 precursors. It plays diverse functions in cellular reprogramming, development, metabolism and tumorigenesis. Many of these functions are executed through its ability to inhibit let-7 maturation. However, little is known about its function independent of let-7. Here we made zebrafish transgenic lines expressing high levels of either wild-type or mutant LIN28B (does not block the maturation of let-7) in the peripheral sympathetic nervous system driven by the dopamine beta-hydroxylase promoter. We bred these lines with a transgenic zebrafish line overexpressing the MYCN oncogene in these cells. Either wild-type or mutant LIN28B overexpression accelerated the onset and increased the penetrance of MYCN-induced neuroblastoma, despite the fact that only wild-type LIN28B blocked let-7 maturation compared to MYCN-only tumors. Mechanistically, both wild-type and mutant LIN28B overexpression enhanced MYCN-induced hyperplasia by increasing cell proliferation in the sympathoadrenal lineage. Further studies revealed that overexpression of either wild-type or mutant LIN28B resulted in hyperphosphorylation of AKT on both Ser473 and Thr308 in zebrafish tumors and also serum-deprived human neuroblastoma cell lines. Both wild-type and mutant LIN28B interacted with IGF2BP1, a known interactor that increases stability and translation of a subset of RNAs. We found IGF2 RNA levels to be increased in response to either form of LIN28B. Coincident with IGF2 overexpression, IGF1R was phosphorylated, providing an explanation for PI3K-AKT activation in LIN28B overexpressing cells. Finally, overexpression of a constitutively active, myristoylated murine Akt2 (myr-mAkt2) alone in zebrafish induced ganglioneuroma in the interregnal gland (the zebrafish equivalent of the human adrenal medulla), without a requirement for MCYN overexpression. Thus, our studies indicate that overexpression of LIN28B leads to AKT activation mediated through its interaction with IGF2BP1 and subsequent upregulation of IGF2. This pathway provides a mechanism underlying enhanced transformation in LIN28B overexpressing neuroblastomas, which is independent of the inhibitory activity of LIN28B on let-7 maturation. Citation Format: Ting Tao, John T. Powers, Hui Shi, Pavlos Missios, Antonio R. Perez-Atayde, Shizhen Zhu, George Q. Daley, Thomas A. Look. LIN28B-mediated let-7 independent activation of AKT promotes neuroblastoma pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1040. doi:10.1158/1538-7445.AM2017-1040

Vascularized lymph node (VLN) transfer has been of high interest in the past decade for the treatment of lymphedema, since it has been shown to be effective in reducing limb volumes, decreasing infectious episodes and improving quality of life. Multiple donor sites have been described in the quest for the optimal one. Herein, we describe a novel lymph node flap option based on the ileocolic artery and vein. The ileocecal vascularized lymph node (IC‐VLN) flap was used in the management of a 33‐year‐old male patient with lower extremity lymphedema secondary to left inguinal trauma. The patient had previously underwent a pedicled omentum flap transposition with minimal improvement in limb size and persistent episodes of infection. At 15 month follow‐up, the IC‐VLN flap improved the lymphatic drainage in the affected limb with a mean limb circumference reduction rate of 26.3%. No donor site complications or further episodes of infection were noted. According to our findings, the IC‐VLN flap may be another option for VLN transfer in very selected cases. Nevertheless, larger series with a longer follow‐up are required to analyze the efficacy and long‐term results of this flap.

The absence of a positive family history (PFH) in 10%–25% of patients poses a diagnostic challenge for autosomal dominant polycystic kidney disease (ADPKD). In the Toronto Genetic Epidemiology Study of Polycystic Kidney Disease, 210 affected probands underwent renal function testing, abdominal imaging, and comprehensive PKD1 and PKD2 mutation screening. From this cohort, we reviewed all patients with and without an apparent family history, examined their parental medical records, and performed renal imaging in all available parents of unknown disease status. Subsequent reclassification of 209 analyzed patients revealed 72.2% (151 of 209) with a PFH, 15.3% (32 of 209) with de novo disease, 10.5% (22 of 209) with an indeterminate family history, and 1.9% (four of 209) with PFH in retrospect. Among the patients with de novo cases, we found two families with germline mosaicism and one family with somatic mosaicism. Additionally, analysis of renal imaging revealed that 16.3% (34 of 209) of patients displayed atypical PKD, most of which followed one of three patterns: asymmetric or focal PKD with PFH and an identified PKD1 or PKD2 mutation (15 of 34), asymmetric and de novo PKD with proven or suspected somatic mosaicism (seven of 34), or focal PKD without any identifiable PKD1 or PKD2 mutation (eight of 34). In conclusion, PKD without an apparent family history may be due to de novo disease, missing parental medical records, germline or somatic mosaicism, or mild disease from hypomorphic PKD1 and PKD2 mutations. Furthermore, mutations of a newly identified gene for ADPKD, GANAB , and somatic mosaicism need to be considered in the mutation-negative patients with focal disease.

Abstract Purpose: Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. Accordingly, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The purpose of this study was to understand whether other AR variants may be coexpressed with AR-V7 and promote resistance to AR-targeted therapies. Experimental Design: We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models. Coexpression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. Expression of AR-V9 protein in CRPC models was evaluated with polyclonal antisera. Multivariate analysis was performed to test whether AR variant mRNA expression in metastatic tissues was associated with a 12-week progression-free survival endpoint in a prospective clinical trial of 78 CRPC-stage patients initiating therapy with the androgen synthesis inhibitor, abiraterone acetate. Results: AR-V9 was frequently coexpressed with AR-V7. Both AR variant species were found to share a common 3′ terminal cryptic exon, which rendered AR-V9 susceptible to experimental manipulations that were previously thought to target AR-V7 uniquely. AR-V9 promoted ligand-independent growth of prostate cancer cells. High AR-V9 mRNA expression in CRPC metastases was predictive of primary resistance to abiraterone acetate (HR = 4.0; 95% confidence interval, 1.31–12.2; P = 0.02). Conclusions: AR-V9 may be an important component of therapeutic resistance in CRPC. Clin Cancer Res; 23(16); 4704–15. ©2017 AACR.

Abstract Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K–AKT–mTOR pathway is associated with specific sites of breast cancer metastasis. Experimental Design: Next-generation sequencing–based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K–AKT–mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K–AKT–mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions. Results: PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined. Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K–AKT–mTOR signaling network. Clin Cancer Res; 23(16); 4919–28. ©2017 AACR.

Prohibitin 1 (PHB1) is best known as a mitochondrial chaperone, and its role in cancer is conflicting. Mice lacking methionine adenosyltransferase α1 (MATα1) have lower PHB1 expression, and we reported that c‐MYC interacts directly with both proteins. Furthermore, c‐MYC and MATα1 exert opposing effects on liver cancer growth, prompting us to examine the interplay between PHB1, MATα1, and c‐MYC and PHB1's role in liver tumorigenesis. We found that PHB1 is highly expressed in normal hepatocytes and bile duct epithelial cells and down‐regulated in most human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). In HCC and CCA cells, PHB1 expression correlates inversely with growth. PHB1 and MAT1A positively regulate each other's expression, whereas PHB1 negatively regulates the expression of c‐MYC, MAFG, and c‐MAF. Both PHB1 and MATα1 heterodimerize with MAX, bind to the E‐box element, and repress E‐box promoter activity. PHB1 promoter contains a repressive E‐box element and is occupied mainly by MAX, MNT, and MATα1 in nonmalignant cholangiocytes and noncancerous tissues that switched to c‐MYC, c‐MAF, and MAFG in cancer cells and human HCC/CCA. All 8‐month‐old liver‐specific Phb1 knockout mice developed HCC, and one developed CCA. Five‐month‐old Phb1 heterozygotes, but not Phb1 flox mice, developed aberrant bile duct proliferation; and one developed CCA 3.5 months after left and median bile duct ligation. Phb1 heterozygotes had a more profound fall in the expression of glutathione synthetic enzymes and higher hepatic oxidative stress following left and median bile duct ligation. Conclusion: We have identified that PHB1, down‐regulated in most human HCC and CCA, heterodimerizes with MAX to repress the E‐box and positively regulates MAT1A while suppressing c‐MYC, MAFG, and c‐MAF expression; in mice, reduced PHB1 expression predisposes to the development of cholestasis‐induced CCA. (Hepatology 2017;65:1249‐1266).

AbstractControl of knee motion in small animal models is necessary to study the effect of mechanical load on the healing process. This can be especially challenging in mice, which are being increasingly used for various orthopedic reconstruction models. We explored the feasibility of botulinum toxin (Botox; Allergan, Dublin, Ireland) paralysis and a newly designed external fixator to restrict motion of the knee in mice undergoing anterior cruciate ligament (ACL) reconstruction. Nineteen C57BL/6 mice were allocated to two groups: (1) Botox group (n = 9) and (2) external fixator group (n = 10). Mice in Botox group received two different doses of Botox: 0.25 unit (n = 3) and 0.5 unit (n = 6). Injection was performed 72 hours prior to ACL reconstruction into the quadriceps, hamstring, and calf muscles of the right hind leg. Mice in external fixator group received an external fixator following ACL reconstruction. Mice were monitored for survival, tolerance, and achievement of complete knee immobilization. All mice were meant for sacrifice on day 14 postoperatively. No perceptible change in gait was observed with 0.25 unit of Botox. All mice that received 0.5 unit of Botox had complete hind limb paralysis documented by footprint analysis 2 days after injection but failed to tolerate anesthesia and were euthanized 24 hours after operation due to their critical condition. In contrast, the external fixator was well tolerated and effectively immobilized the limb. There was a single occurrence of intraoperative technical error in the external fixator group that led to euthanasia. No mechanical failure or complication was observed. Botox paralysis was not a viable option for postoperative restriction of motion and joint loading in mice. However, external fixation was an effective method for complete knee immobilization and can be used in murine models requiring postoperative control of knee loading. This study introduces a robust research tool to allow control of postoperative joint loading in animal models such as ACL reconstruction, permitting study of the effects of mechanical load on the biologic aspects of tendon-to-bone healing.

Introduction: The therapeutic trough range for mexiletine (0.8–2 mcg/mL) was largely established in the setting of arrhythmia prophylaxis following myocardial infarction. Objective: Describe the usage patterns of serum mexiletine concentrations and the impact of these concentrations on mexiletine dosing in modern practice for ventricular arrhythmia treatment. Methods: A single-center, retrospective analysis was conducted using the electronic medical record to identify serum mexiletine concentrations drawn between December 2004 and December 2014. The primary endpoint was the incidence of mexiletine concentrations drawn as troughs. Secondary outcomes included the incidence of mexiletine concentrations that prompted a dose change, association between adverse events and elevated concentrations, and association between baseline characteristics and mexiletine concentrations. Results: A total of 237 individual concentrations were included for analysis with 109 (46.0%) drawn appropriately as trough concentrations. Only 31 (13.1%) of the 237 concentrations drawn prompted a dose change. Mexiletine was primarily used for the treatment of ventricular arrhythmias (96.2%), and 108 (45.6%) concentrations were drawn in an effort to assess efficacy. The median concentration was statistically different between patients with and without an adverse event (0.8 vs 0.7 mcg/mL, respectively; p = 0.017), but may not represent a clinical significance. Patients with hepatic dysfunction had higher median concentrations compared to those without hepatic dysfunction (1.30 vs 1.07 mcg/mL; p = 0.01). Conclusion: Mexiletine concentrations are often drawn at inappropriate times and seldom influence a dose change. This study suggests that routine monitoring of mexiletine concentrations may not be necessary; however, therapeutic drug monitoring may be considered in patients with hepatic dysfunction or to confirm mexiletine absorption in patients where this represents a concern.

Abstract Hepatocellular carcinoma is one of the most lethal cancers in the world. Most of the patients are diagnosed at advanced stage, and they receive sorafenib as their treatment modality. However, sorafenib can only extend patients’ survival to a median of 3-4 months. Hence, there is an urgent need to identify a new therapeutic target for treatment of this disease. Increasing evidence showed that tumor-initiating cells (T-ICs) are intrinsically resistant to conventional treatments. Identification of the signalling pathways that maintain the functions of T-ICs provides potential targets for treatment of HCC. For this purpose, we cultured serial passages of hepatospheres combined with chemotherapeutic regimens as a strategy to enrich liver T-IC population. Using cDNA microarray, we found upregulation of several key enzymes in lipogenesis in enriched T-IC population, among which Stearoyl CoA desaturase-1 (SCD1), the enzyme involved in the conversion of saturated into monounsaturated fatty acids, was the most significant. In HCC clinical samples, 62% (36/58) was found to be overexpressed (> 2-fold) when compared with non-tumor counterparts and patients with SCD1 overexpression had shorter disease free survival. Using overexpression and knockdown approaches, SCD1 was found to regulate the traits of T-ICs, including tumorigenicity, self-renewal, drug resistance and expression of liver T-IC markers. SCD1 was found critical in survival of T-ICs, as enriched T-IC population are more sensitive to the inhibition of SCD1 compared to adherent lineages. Interestingly, SCD1 was found to be upregulated in sorafenib-resistant HCC cells. Pharmacological inhibition of SCD1 by A939572 not only suppressed self-renewal ability but also consistently enhanced the sensitivity towards sorafenib. Using a patient-derived xenograft model, we found that a new inhibitor against SCD1 demonstrated significant growth suppressive effect. By comparing the genetic profiles between SCD1 knockdown cells and control cells by RNA sequencing analysis, we found that upregulation of ER stress signature genes may be the potential downstream targets of SCD1 to maintain the properties of T-ICs. In conclusion, we demonstrated the crucial role of SCD1 in maintenance of liver T-ICs, and targeting SCD1 in combination with sorafenib might be a novel therapeutic regimen against HCC. Citation Format: Kin Fai Ma, Jessica Lo, Eunice Yuen-Ting Lau, John A. Copland, Irene Oi-Lin Ng, Terence Kin-Wah Lee. Targeting liver-tumor initiating cells via hampering the lipogenesis pathways through stearoyl - CoA desaturase. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1003.