p90RSK pathway inhibition synergizes with cisplatin in TMEM16A overexpressing head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) constitutes one of the most common types of human cancers and often metastasizes to lymph nodes. Platinum-based chemotherapeutic drugs are commonly used for treatment of a wide range of cancers, including HNSCC. Its mode of action relies on its ability to impede DNA repair mechanisms, inducing apoptosis in cancer cells. However, due to acquired resistance and toxic side-effects, researchers have been focusing on developing novel combinational therapeutic strategies to overcome cisplatin resistance. In the current study, we identified p90RSK, an ERK1/2 downstream target, as a key mediator and a targetable signaling node against cisplatin resistance. Our results strongly support the role of p90RSK in cisplatin resistance and identify the combination of p90RSK inhibitor, BI-D1870, with cisplatin as a novel therapeutic strategy to overcome cisplatin resistance. In addition, we have identified TMEM16A expression as a potential upstream regulator of p90RSK through the ERK pathway and a biomarker of response to p90RSK targeted therapy in the context of cisplatin resistance.

Targeting the EMT transcription factor TWIST1 overcomes resistance to EGFR inhibitors in EGFR-mutant non-small-cell lung cancer.

Patients with EGFR-mutant non-small-cell lung cancer (NSCLC) have significantly benefited from the use of EGFR tyrosine kinase inhibitors (TKIs). However, long-term efficacy of these therapies is limited due to de novo resistance (~30%) as well as acquired resistance. Epithelial-mesenchymal transition transcription factors (EMT-TFs), have been identified as drivers of EMT-mediated resistance to EGFR TKIs, however, strategies to target EMT-TFs are lacking. As the third generation EGFR TKI, osimertinib, has now been adopted in the first-line setting, the frequency of T790M mutations will significantly decrease in the acquired resistance setting. Previously less common mechanisms of acquired resistance to first generation EGFR TKIs including EMT are now being observed at an increased frequency after osimertinib. Importantly, there are no other FDA approved targeted therapies after progression on osimertinib. Here, we investigated a novel strategy to overcome EGFR TKI resistance through targeting the EMT-TF, TWIST1, in EGFR-mutant NSCLC. We demonstrated that genetic silencing of TWIST1 or treatment with the TWIST1 inhibitor, harmine, resulted in growth inhibition and apoptosis in EGFR-mutant NSCLC. TWIST1 overexpression resulted in erlotinib and osimertinib resistance in EGFR-mutant NSCLC cells. Conversely, genetic and pharmacological inhibition of TWIST1 in EGFR TKI-resistant EGFR-mutant cells increased sensitivity to EGFR TKIs. TWIST1-mediated EGFR TKI resistance was due in part to TWIST1 suppression of transcription of the pro-apoptotic BH3-only gene, BCL2L11 (BIM), by directly binding to BCL2L11 intronic regions and promoter. As such, pan-BCL2 inhibitor treatment overcame TWIST1-mediated EGFR TKI resistance and were more effective in the setting of TWIST1 overexpression. Finally, in a mouse model of autochthonous EGFR-mutant lung cancer, Twist1 overexpression resulted in erlotinib resistance and suppression of erlotinib-induced apoptosis. These studies establish TWIST1 as a driver of resistance to EGFR TKIs and provide rationale for use of TWIST1 inhibitors or BCL2 inhibitors as means to overcome EMT-mediated resistance to EGFR TKIs.

HSP90 inhibition targets autophagy and induces a CASP9-dependent resistance mechanism in NSCLC.

Macroautophagy/autophagy has emerged as a resistance mechanism to anticancer drug treatments that induce metabolic stress. Certain tumors, including a subset of KRAS-mutant NSCLCs have been shown to be addicted to autophagy, and potentially vulnerable to autophagy inhibition. Currently, autophagy inhibition is being tested in the clinic as a therapeutic component for tumors that utilize this degradation process as a drug resistance mechanism. The current study provides evidence that HSP90 (heat shock protein 90) inhibition diminishes the expression of ATG7, thereby impeding the cellular capability of mounting an effective autophagic response in NSCLC cells. Additionally, an elevation in the expression level of CASP9 (caspase 9) prodomain in KRAS-mutant NSCLC cells surviving HSP90 inhibition appears to serve as a cell survival mechanism. Initial characterization of this survival mechanism suggests that the altered expression of CASP9 is mainly ATG7 independent; it does not involve the apoptotic activity of CASP9; and it localizes to a late endosomal and pre-lysosomal phase of the degradation cascade. HSP90 inhibitors are identified here as a pharmacological approach for targeting autophagy via destabilization of ATG7, while an induced expression of CASP9, but not its apoptotic activity, is identified as a resistance mechanism to the cellular stress brought about by HSP90 inhibition.

Targeting Heat Shock Proteins in Cancer: A Promising Therapeutic Approach.

Heat shock proteins (HSPs) are a large family of chaperones that are involved in protein folding and maturation of a variety of "client" proteins protecting them from degradation, oxidative stress, hypoxia, and thermal stress. Hence, they are significant regulators of cellular proliferation, differentiation and strongly implicated in the molecular orchestration of cancer development and progression as many of their clients are well established oncoproteins in multiple tumor types. Interestingly, tumor cells are more HSP chaperonage-dependent than normal cells for proliferation and survival because the oncoproteins in cancer cells are often misfolded and require augmented chaperonage activity for correction. This led to the development of several inhibitors of HSP90 and other HSPs that have shown promise both preclinically and clinically in the treatment of cancer. In this article, we comprehensively review the roles of some of the important HSPs in cancer, and how targeting them could be efficacious, especially when traditional cancer therapies fail.

A First-in-Class TWIST1 Inhibitor with Activity in Oncogene-Driven Lung Cancer.

TWIST1, an epithelial-mesenchymal transition (EMT) transcription factor, is critical for oncogene-driven non-small cell lung cancer (NSCLC) tumorigenesis. Given the potential of TWIST1 as a therapeutic target, a chemical-bioinformatic approach using connectivity mapping (CMAP) analysis was used to identify TWIST1 inhibitors. Characterization of the top ranked candidates from the unbiased screen revealed that harmine, a harmala alkaloid, inhibited multiple TWIST1 functions, including single-cell dissemination, suppression of normal branching in 3D epithelial culture, and proliferation of oncogene driver-defined NSCLC cells. Harmine treatment phenocopied genetic loss of TWIST1 by inducing oncogene-induced senescence or apoptosis. Mechanistic investigation revealed that harmine targeted the TWIST1 pathway through its promotion of TWIST1 protein degradation. As dimerization is critical for TWIST1 function and stability, the effect of harmine on specific TWIST1 dimers was examined. TWIST1 and its dimer partners, the E2A proteins, which were found to be required for TWIST1-mediated functions, regulated the stability of the other heterodimeric partner posttranslationally. Harmine preferentially promoted degradation of the TWIST1-E2A heterodimer compared with the TWIST-TWIST1 homodimer, and targeting the TWIST1-E2A heterodimer was required for harmine cytotoxicity. Finally, harmine had activity in both transgenic and patient-derived xenograft mouse models of KRAS-mutant NSCLC. These studies identified harmine as a first-in-class TWIST1 inhibitor with marked anti-tumor activity in oncogene-driven NSCLC including EGFR mutant, KRAS mutant and MET altered NSCLC.Implications: TWIST1 is required for oncogene-driven NSCLC tumorigenesis and EMT; thus, harmine and its analogues/derivatives represent a novel therapeutic strategy to treat oncogene-driven NSCLC as well as other solid tumor malignancies. Mol Cancer Res; 15(12); 1764-76. ©2017 AACR.

Reactivation of the p90RSK-CDC25C Pathway Leads to Bypass of the Ganetespib-Induced G2-M Arrest and Mediates Acquired Resistance to Ganetespib in KRAS-Mutant NSCLC.

A subset of non-small cell lung cancers (NSCLC) are dependent upon oncogenic driver mutations, including the most frequently observed driver mutant KRAS, which is associated with a poor prognosis. As direct RAS targeting in the clinic has been unsuccessful to date, use of Hsp90 inhibitors appeared to be a promising therapy for KRAS-mutant NSCLC; however, limited clinical efficacy was observed due to rapid resistance. Furthermore, the combination of the Hsp90 inhibitor (Hsp90i), ganetespib, and docetaxel was tested in a phase III clinical trial and failed to demonstrate benefit. Here, we investigated the mechanism(s) of resistance to ganetespib and explored why the combination with docetaxel failed in the clinic. We have not only identified a critical role for the bypass of the G2-M cell-cycle checkpoint as a mechanism of ganetespib resistance (GR) but have also found that GR leads to cross-resistance to docetaxel. Reactivation of p90RSK and its downstream target, CDC25C, was critical for GR and mediated the bypass of a G2-M arrest. Overexpression of either p90RSK or CDC25C lead to bypass of G2-M arrest and induced ganetespib resistance in vitro and in vivo Moreover, resistance was dependent on p90RSK/CDC25C signaling, as synthetic lethality to ERK1/2, p90RSK, or CDC25C inhibitors was observed. Importantly, the combination of ganetespib and p90RSK or CDC25C inhibitors was highly efficacious in parental cells. These studies provide a way forward for Hsp90 inhibitors through the development of novel rationally designed Hsp90 inhibitor combinations that may prevent or overcome resistance to Hsp90i. Mol Cancer Ther; 16(8); 1658-68. ©2017 AACR.

Acquired Resistance to the Hsp90 Inhibitor, Ganetespib, in KRAS-Mutant NSCLC Is Mediated via Reactivation of the ERK-p90RSK-mTOR Signaling Network.

Approximately 25% of non-small cell lung cancer (NSCLC) patients have KRAS mutations, and no effective therapeutic strategy exists for these patients. The use of Hsp90 inhibitors in KRAS-mutant NSCLC appeared to be a promising approach, as these inhibitors target many KRAS downstream effectors; however, limited clinical efficacy has been observed due to resistance. Here, we examined the mechanism(s) of acquired resistance to the Hsp90 inhibitor, ganetespib, and identified novel and rationally devised Hsp90 inhibitor combinations, which may prevent and overcome resistance to Hsp90 inhibitors. We derived KRAS-mutant NSCLC ganetespib-resistant cell lines to identify the resistance mechanism(s) and identified hyperactivation of RAF/MEK/ERK/RSK and PI3K/AKT/mTOR pathways as key resistance mechanisms. Furthermore, we found that ganetespib-resistant cells are "addicted" to these pathways, as ganetespib resistance leads to synthetic lethality to a dual PI3K/mTOR, a PI3K, or an ERK inhibitor. Interestingly, the levels and activity of a key activator of the mTOR pathway and an ERK downstream target, p90 ribosomal S6 kinase (RSK), were also increased in the ganetespib-resistant cells. Genetic or pharmacologic inhibition of p90RSK in ganetespib-resistant cells restored sensitivity to ganetespib, whereas p90RSK overexpression induced ganetespib resistance in naïve cells, validating p90RSK as a mediator of resistance and a novel therapeutic target. Our studies offer a way forward for Hsp90 inhibitors through the rational design of Hsp90 inhibitor combinations that may prevent and/or overcome resistance to Hsp90 inhibitors, providing an effective therapeutic strategy for KRAS-mutant NSCLC. Mol Cancer Ther; 16(5); 793-804. ©2017 AACR.

HSP90 inhibitors in lung cancer: promise still unfulfilled.

Despite recent advances in the treatment of lung cancer, non-small cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths in the United States and worldwide, with a 5-year survival rate of less than 17%. Analysis of the molecular drivers of NSCLC led to the recognition that NSCLC is a collection of distinct, molecularly driven neoplasms. Several subsets of NSCLC with clinical relevance to targeted therapies are defined based on alterations in EGFR, ALK, and other key oncogenic drivers. However, for many oncogenic drivers-such as mutant KRAS-targeted therapies are lacking. Heat shock protein 90 (HSP90) is an adenosine triphosphate (ATP)-dependent molecular chaperone that is critically required for the stability of its clientele, many of which are driver oncoproteins. Therefore, HSP90 inhibitors could prove to be an effective and alternate approach to treat patients with NSCLC that has a specific molecular background or that has acquired resistance to other drugs. Over the last 2 decades, several HSP90 inhibitors have been developed that produced promising preclinical and clinical results. The quest is far from over, however. In this review, we discuss the development and the preclinical and clinical profiles of some of the HSP90 inhibitors that may help to improve the targeted treatment of NSCLC.

Spondyloepiphyseal dysplasia tarda with progressive arthropathy associated with subcapsular cataract.

Spondyloepiphyseal dysplasia tarda with progressive arthropathy is a form of inherited skeletal dysplasia involving the axial skeleton along with swelling and deformities of the peripheral joints that mimics juvenile rheumatoid arthritis. We report a case of a 14-year-old school boy who presented with dwarfism, spine deformity, ocular anomaly and peripheral arthropathy with characteristic radiological changes in the vertebrae and multiple other joints.

Positive surgical margins at radical prostatectomy: Population-based averages within PSA and Gleason strata.

BACKGROUND: Positive surgical margins (PSM) are an important determinant of biochemical recurrence after radical prostatectomy (RP). We use a population-based cancer registry to evaluate PSM by stage, Gleason and prostate-specific antigen (PSA). METHODS: We identified men undergoing RP from the Surveillance, Epidemiology and End Results (SEER) database between 2004 and 2007. Differences between those with and without PSM were compared with chi-squared tests. The proportion of cases with PSM were stratified by PSA and Gleason sum for both pT2 and pT3a tumours. Factors associated with PSM were analyzed using chi square and multivariate logistic regression analysis. A composite variable was used in a second multivariate analysis to display the odds ratio (OR) for a PSM for each discrete combination of PSA, Gleason score and pT stage. RESULTS: In total, 28 461 RP patients were identified and a PSM was present in 19.5%. PSM were 42% in pT3a and 16% in pT2 cases. Higher PSAs (<4.0, 4-9.9, >10) were associated with higher proportions of PSM (12%, 20% and 28%, p < 0.001). Similarly, higher Gleason scores (≤6, 3+4, 4+3, ≥8) were associated with higher PSM (12%, 22%, 27% and 33%, p < 0.001). For pT2 tumours, the proportion of PSM ranged from 8% (Gleason ≤6, PSA <4.0) to 28% (Gleason 8-10, PSA ≥10). For pT3a tumours, the PSM was higher in each Gleason/PSA strata compared to those with pT2 tumours, reaching 63% for those with pT3a, Gleason 8-10, PSA >10 disease. On multivariate analysis, stage was the largest predictor for PSM (OR 3.05, 95% confidence interval 2.81-3.30), although Gleason score and PSA remained statistically significant. CONCLUSION: In this population-based study of PSM after RP, the proportion of PSM vary significantly within different PSA and Gleason strata for organ-confined and extracapsular disease. These data can be used as a reference for urologist self-assessment.

Transfer of laparoscopic radical prostatectomy skills from bench model to animal model: a prospective, single-blind, randomized, controlled study.

PURPOSE: Learning laparoscopic urethrovesical anastomosis is a crucial step in laparoscopic radical prostatectomy. Previously we noted that practice on a low fidelity urethrovesical model was more effective for trainees than basic suturing drills on a foam pad when learning laparoscopic urethrovesical anastomosis skills. We evaluated learner transfer of skills, specifically whether skills learned on the urethrovesical model would transfer to a high fidelity, live animal model. MATERIALS AND METHODS: A total of 28 senior residents, fellows and staff surgeons in urology, general surgery and gynecology were randomized to 2 hours of laparoscopic urethrovesical anastomosis training on a urethrovesical model (group 1) or to basic laparoscopic suturing and knot tying on foam pads (group 2). All participants then performed timed laparoscopic urethrovesical anastomosis on anesthetized female pigs. A blinded urologist scored subject videotaped performance using checklist, global rating scale and end product rating scores. RESULTS: Group 1 was significantly more adept than group 2 at the laparoscopic urethrovesical anastomosis pig task when measured by the checklist, global rating scale and end product rating (each p <0.05). Time to completion was similar in the 2 groups. No statistically significant difference was noted in global rating scale and checklist scores for laparoscopic urethrovesical anastomosis performed on the urethrovesical model vs the pig. CONCLUSIONS: Training on a urethrovesical model is superior to training with basic laparoscopic suturing on a foam pad for performing laparoscopic urethrovesical anastomosis skills on an anesthetized female pig. Skills learned on a urethrovesical model transfer to a high fidelity, live animal model.

Renal histopathology features according to various warm ischemia times in porcine laparoscopic and open surgery model.

BACKGROUND: Thirty minutes has been considered as the threshold for tolerable warm ischemic time (WIT). Recent reports demonstrate recovery of renal function after longer WIT. We assessed renal histology according to different WIT in a 2-kidney porcine model. METHODS: Twelve female pigs were randomized to an open or laparoscopic group. Each pig was further randomized within each group to clamping the left renal artery for 5, 15, 30, 45, 60 or 180 minutes. Preclamping left renal biopsies were performed on each pig. The contralateral kidney in each animal was used as an individual control. On postoperative day 14, all animals underwent bilateral nephrectomies. Preclamping left renal biopsies and all renal specimens were evaluated by a blinded veterinary pathologist. RESULTS: One pig died in the open group after 180 minutes of clamping. Histopathology did not show any significant changes between the two groups and across clamp times from 5 to 60 minutes. After 180 minutes of laparoscopic clamping, there was evidence of diffuse necrosis. INTERPRETATION: Sixty minutes of ischemia did not show any permanent renal damage in both groups. Further studies are needed to verify these findings in humans. A prolonged ischemic time without permanent renal damage would be helpful in partial nephrectomy. Warm ischemic time of 180 minutes exceeded the renal ischemic burden based on histological features.

Assessing the surgical decision making abilities of novice and proficient urologists.

PURPOSE: We examined whether the Surgical Decision Making Rating Scale can measure a difference in surgical judgment among urologists at various levels of training. MATERIALS AND METHODS: A total of 25 medical students, urology residents and staff urologists viewed clips from 8 select urological procedures and verbalized their thought processes. The clips were ordered in increasing complexity from lower level tasks (catheterization and cystoscopy) to more advanced procedures (laser lithotripsy, and open and laparoscopic prostatectomy and nephrectomy). Performance was transcribed and blindly rated using the previously validated rating scale. Subjects were also asked to self-evaluate their performance using this scale. RESULTS: Overall the rating scale distinguished the training level across knowledge domains (anatomy and management of the current task) and judgment domains (avoiding complications, higher reasoning and immediate surgical planning). The mean score across all training levels was 112 of 200 (range 51 to 161). Scale performance showed a significant correlation with seniority (rho = 0.96, p <0.05). This trend persisted when performance was analyzed separately for knowledge and judgment domain elements (rho = 0.95 and 0.96, respectively, each p <0.05). Self-evaluation correlated well with blinded evaluation across all levels of training (rho = 0.87, p = 0.01). CONCLUSIONS: The Surgical Decision Making Rating Scale can reliably detect differences in knowledge and surgical judgment among medical students, urology residents and staff urologists. This tool has potential applications for evaluating trainees and determining subjects with proficient decision making abilities. It also shows a significant correlation between self-rated performance and blinded evaluation.

Lower plasma adiponectin levels are associated with larger tumor size and metastasis in clear-cell carcinoma of the kidney.

OBJECTIVES: To examine a possible relationship between plasma adiponectin levels and renal cell carcinoma (RCC). Adiponectin, a cytokine secreted by adipocytes, is a potent antiangiogenic factor. Plasma levels of adiponectin in patients with RCC and tumor adiponectin receptors R1 and R2 (AdipoR1&2) expression levels were measured and correlated with disease characteristics. METHODS: Preoperative plasma samples from 42 patients were analyzed in triplicate for adiponectin levels with a specific ELISA assay. All patients had clear-cell RCC, including 15 with metastatic disease. Diabetic patients were excluded; all had normal renal function. The RCC and surrounding normal renal tissue were comparatively analyzed for AdipoR1&2 expressions (immunoblotting) in 15 patients. RESULTS: Mean, median, and range of plasma adiponectin levels were 6.33, 5.84, and 1-25.2 microg/ml, respectively. A strong inverse correlation was found between plasma adiponectin levels and tumor size with significantly lower levels of adiponectin in tumors > or =4 cm (p<0.01). The median adiponectin levels in metastatic and nonmetastatic patients were 4.08 and 7.4 microg/ml, respectively (p=0.029). A trend toward significant lower adiponectin levels in high versus low Fuhrman grade (3 and 4 vs. 1 and 2) was noted (p=0.057). Expression of AdipoR1&R2 was found to be lower in tumor tissue compared with the patient's normal surrounding kidney tissues in 40% of the cases. Metastatic tumors expressed lower levels of AdipoR2. Body mass index was not inversely correlated with adiponectin levels. CONCLUSIONS: Lower blood levels of adiponectin are positively associated with clear-cell RCC aggressiveness and could potentially be used as a biomarker.

Durability of endourologic skills: two-year follow-up study.

PURPOSE: To assess the long-term durability of endourologic skills among urology trainees after an intensive technical skills training course. SUBJECTS AND METHODS: Seventeen urology residents participated in a 2-day ureteroscopy course at a surgical skills center. Residents performed rigid ureteroscopy and basket manipulation of a small midureteral stone. Performance was assessed immediately after the course and 1 year and 2 years after training. Residents prospectively tracked all ureteroscopic cases in which they were considered the primary surgeon (i.e., performed greater than 75% of the procedure). Performance was measured using a validated global rating score (GRS), checklist score (CLS), and time required to complete the task. RESULTS: Overall, GRS improved over the 2-year follow-up (P < 0.001), with most of the improvement occurring in the first year (P = 0.03). The CLS and time to complete the task did not change (P = 0.08 and 0.12, respectively). At the 2-year follow-up, the number of cases logged had no significant effect on performance. CONCLUSIONS: Ureteroscopy skills are retained and continue to improve 2 years after completing an intense training session that uses high-fidelity bench models. Ureteroscopic experience is important for the maintenance and development of skills, even though they appear to plateau after 1 year. This result may also reflect a ceiling effect of the assessment tools.