Michael J. Pishvaian

Cross-regulation of β-catenin–LEF/TCF and retinoid signaling pathways

Abstract Vitamin A derivatives (retinoids) are potent regulators of embryogenesis, cell proliferation, epithelial cell differentiation and carcinogenesis [1]. In breast cancer cells, the effects of retinoids are associated with changes in the cadherin–β-catenin adhesion and signaling system [2,3]. β-catenin is a component of the Wnt signaling pathway, which regulates several developmental pathways [4]. Increases in cytoplasmic β-catenin and β-catenin signaling are also associated with numerous cancers, and are particularly important in colon cancer [5]. The oncogenic and developmental effects of β-catenin are mediated by its interaction with and activation of members of the LEF/TCF family of transcription factors [6–8]. Here, we shown that retinoic acid (RA) decreases the activity of the β-catenin–LEF/TCF signaling pathway. This activity of RA was independent of the adenomatous polyposis coli (APC) tumor suppressor and ubiquitination-dependent degradation of cytoplasmic β-catenin. Consistent with this finding, β-catenin interacted directly with the RA receptor (RAR) in a retinoid-dependent manner, but not with the retinoid X receptor (RXR), and RAR competed with TCF for β-catenin binding. The activity of RA on RAR-responsive promoters was also potentiated by β-catenin. The data suggest that direct regulation of β-catenin–LEF/TCF signaling is one mechanism whereby RA influences development, cell differentiation and cancer.

Disruption of transforming growth factor-β signaling through β-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation

Transforming growth factor-β (TGF-β) signaling members, TGF-β receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf+/− mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P<0.017). ELF and TBRII are also markedly decreased in human HCC cell lines SNU-398 and SNU-475. Restoration of ELF and TBRII in SNU-398 cells markedly decreases cyclin D1 as well as hyperphosphorylated-retinoblastoma (hyperphosphorylated-pRb). Thus, we show that TGF-β signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.

Stereotactic body radiation therapy with concurrent full-dose gemcitabine for locally advanced pancreatic cancer: a pilot trial demonstrating safety.

BackgroundConcurrent chemoradiation is a standard option for locally advanced pancreatic cancer (LAPC). Concurrent conventional radiation with full-dose gemcitabine has significant toxicity. Stereotactic body radiation therapy (SBRT) may provide the opportunity to administer radiation in a shorter time frame with similar efficacy and reduced toxicity. This Pilot study assessed the safety of concurrent full-dose gemcitabine with SBRT for LAPC.MethodsPatients received gemcitabine, 1000 mg/m2 for 6 cycles. During week 4 of cycle 1, patients received SBRT (25 Gy delivered in five consecutive daily fractions of 5 Gy prescribed to the 75-83% isodose line). Acute and late toxicities were assessed using NIH CTCAE v3. Tumor response was assessed by RECIST. Patients underwent an esophagogastroduodenoscopy at baseline, 2, and 6 months to assess the duodenal mucosa. Quality of life (QoL) data was collected before and after treatment using the QLQ-C30 and QLQ-PAN26 questionnaires.ResultsBetween September 2009 and February 2011, 11 patients enrolled with one withdrawal during radiation therapy. Patients had grade 1 to 2 gastrointestinal toxicity from the start of SBRT to 2 weeks after treatment. There were no grade 3 or greater radiation-related toxicities or delays for cycle 2 of gemcitabine. On endoscopy, there were no grade 2 or higher mucosal toxicities. Two patients had a partial response. The median progression free and overall survival were 6.8 and 12.2 months, respectively. Global QoL did not change between baseline and immediately after radiation treatment.ConclusionsSBRT with concurrent full dose gemcitabine is safe when administered to patients with LAPC. There is no delay in administration of radiation or chemotherapy, and radiation is completed with minimal toxicity.

Safety and Antitumor Activity of Pembrolizumab in Advanced Programmed Death Ligand 1–Positive Endometrial Cancer: Results From the KEYNOTE-028 Study

Purpose The multicohort phase Ib KEYNOTE-028 (NCT02054806) study was designed to evaluate the safety and efficacy of pembrolizumab, an anti-programmed death 1 monoclonal antibody, in patients with programmed death ligand 1 (PD-L1) -positive advanced solid tumors. The results from the advanced endometrial cancer cohort are reported. Patients and Methods Female patients with locally advanced or metastatic PD-L1-positive endometrial cancer who had experienced progression after standard therapy were eligible. Patients received pembrolizumab 10 mg/kg every 2 weeks for up to 24 months or until progression or unacceptable toxicity. Primary efficacy end point was objective response rate by RECIST (version 1.1). Secondary end points included safety, duration of response (DOR), progression-free survival, and overall survival. The data cutoff was February 17, 2016. Results Of 75 patients screened, 36 (48.0%) had PD-L1-positive tumors, and 24 (32.0%) were enrolled. Fifteen (62.5%) of these 24 patients had received at least two previous lines of therapy for advanced disease. Three patients (13.0%) achieved confirmed partial response (95% CI, 2.8% to 33.6%); the median DOR was not reached. Two patients were still receiving treatment and exhibiting continued response at time of data cutoff. Three additional patients (13.0%) achieved stable disease, with a median duration of 24.6 weeks. One patient who achieved partial response had a polymerase E mutation. Thirteen patients (54.2%) experienced treatment-related adverse events (AEs), with fatigue (20.8%), pruritus (16.7%), pyrexia (12.5%), and decreased appetite (12.5%) occurring in ≥ 10% of patients. Grade 3 treatment-related AEs were reported in four patients. No patient experienced a grade 4 AE, and no patient discontinued treatment because of an AE. Conclusion Pembrolizumab demonstrated a favorable safety profile and durable antitumor activity in a subgroup of patients with heavily pretreated advanced PD-L1-positive endometrial cancer.

A Phase 1 Study of Efatutazone, an Oral Peroxisome Proliferator-Activated Receptor Gamma Agonist, Administered to Patients With Advanced Malignancies

Efatutazone (CS‐7017), a novel peroxisome proliferator‐activated receptor gamma (PPARγ) agonist, exerts anticancer activity in preclinical models. The authors conducted a phase 1 study to determine the recommended phase 2 dose, safety, tolerability, and pharmacokinetics of efatutazone.

Transforming growth factor-β adaptor, β2-spectrin, modulates cyclin dependent kinase 4 to reduce development of hepatocellular cancer†

Transforming growth factor beta (TGF‐β) is an important regulator of cell growth, and loss of TGF‐β signaling is a hallmark of carcinogenesis. The Smad3/4 adaptor protein β2‐spectrin (β2SP) is emerging as a potent regulator of tumorigenesis through its ability to modulate the tumor suppressor function of TGF‐β. However, to date the role of the TGF‐β signaling pathway at specific stages of the development of hepatocellular carcinoma (HCC), particularly in relation to the activation of other oncogenic pathways, remains poorly delineated. Here we identify a mechanism by which β2SP, a crucial Smad3 adaptor, modulates cyclin dependent kinase 4 (CDK4), cell cycle progression, and suppression of HCC. Increased expression of β2SP inhibits phosphorylation of the retinoblastoma gene product (Rb) and markedly reduces CDK4 expression to a far greater extent than other CDKs and cyclins. Furthermore, suppression of CDK4 by β2SP efficiently restores Rb hypophosphorylation and cell cycle arrest in G1. We further demonstrate that β2SP interacts with CDK4 and Smad3 in a competitive and TGF‐β‐dependent manner. In addition, haploinsufficiency of cdk4 in β2sp+/− mice results in a dramatic decline in HCC formation compared to that observed in β2sp+/− mice. Conclusion: β2SP deficiency leads to CDK4 activation and contributes to dysregulation of the cell cycle, cellular proliferation, oncogene overexpression, and the formation of HCCs. Our data highlight CDK4 as an attractive target for the pharmacologic inhibition of HCC and demonstrate the importance of β2sp+/− mice as a model of preclinical efficacy in the treatment of HCC. (HEPATOLOGY 2011;)

Restoration of DNA-binding and growth-suppressive activity of mutant forms of p53 via a PCAF-mediated acetylation pathway†

Tumor‐derived mutant forms of p53 compromise its DNA binding, transcriptional, and growth regulatory activity in a manner that is dependent upon the cell‐type and the type of mutation. Given the high frequency of p53 mutations in human tumors, reactivation of the p53 pathway has been widely proposed as beneficial for cancer therapy. In support of this possibility p53 mutants possess a certain degree of conformational flexibility that allows for re‐induction of function by a number of structurally different artificial compounds or by short peptides. This raises the question of whether physiological pathways for p53 mutant reactivation also exist and can be exploited therapeutically. The activity of wild‐type p53 is modulated by various acetyl‐transferases and deacetylases, but whether acetylation influences signaling by p53 mutant is still unknown. Here, we show that the PCAF acetyl‐transferase is down‐regulated in tumors harboring p53 mutants, where its re‐expression leads to p53 acetylation and to cell death. Furthermore, acetylation restores the DNA‐binding ability of p53 mutants in vitro and expression of PCAF, or treatment with deacetylase inhibitors, promotes their binding to p53‐regulated promoters and transcriptional activity in vivo. These data suggest that PCAF‐mediated acetylation rescues activity of at least a set of p53 mutations. Therefore, we propose that dis‐regulation of PCAF activity is a pre‐requisite for p53 mutant loss of function and for the oncogenic potential acquired by neoplastic cells expressing these proteins. Our findings offer a new rationale for therapeutic targeting of PCAF activity in tumors harboring oncogenic versions of p53. J. Cell. Physiol. 225: 394–405, 2010.

Biomarkers of WNT signaling

WNT signaling plays a key role in the developing embryo and in the maintenance of a stem/progenitor cell compartment in adult tissues. However, WNT signaling is also believed to play an important role in carcinogenesis. WNT signaling may be activated directly through WNT ligand overexpression, or more often through mutations downstream in the WNT signaling cascade. The common endpoint is an inappropriate expansion of a stem cell compartment and proliferation of more differentiated daughter cells which ultimately acquire additional mutations leading to the development of a malignant, invasive cancer cell. It is likely that WNT signaling is at the core of many human cancers, but no definitive biomarker of WNT activity has been established. Furthermore, while therapies targeting WNT-responsive gene products such as COX-2 and VEGF have been developed, no therapy directly targeting WNT signaling itself has yet made it into the clinic. We present here a brief summary of the WNT signaling cascade, and focus particularly on markers of WNT activity that have diagnostic, prognostic, or therapeutic relevance. Finally, we propose the development of novel ligands of the steroid hormone family of nuclear receptors to be used as therapies that specifically target and inhibit WNT/beta-catenin/TCF-mediated transcriptional activity.

Irreversible Multitargeted ErbB Family Inhibitors for Therapy of Lung and Breast Cancer

Overactivation of the ErbB protein family, which is comprised of 4 receptor tyrosine kinase members (ErbB1/epidermal growth factor receptor [EGFR]/HER1, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), can drive the development and progression of a wide variety of malignancies, including colorectal, head and neck, and certain non-small cell lung cancers (NSCLCs). As a result, agents that target a specific member of the ErbB family have been developed for the treatment of cancer. These agents include the reversible EGFR tyrosine kinase inhibitors (TKIs) erlotinib and gefitinib; the EGFR-targeting monoclonal antibodies cetuximab and panitumumab; and the HER2-targeting monoclonal antibody trastuzumab. Lapatinib is a dual TKI that targets both EGFR and HER2. In addition, TKIs that inhibit multiple members of the ErbB family and also bind their targets irreversibly are under evaluation for the treatment of cancer. Three such compounds have progressed into clinical studies: the EGFR, HER2, and HER4 inhibitors afatinib, dacomitinib, and neratinib. Phase I studies of these agents have shown clinical activity in NSCLC, breast cancer, and other malignancies. Currently, afatinib is approved for EGFR mutation-positive NSCLC and is in development for squamous NSCLC, and dacomitinib is in phase III of clinical development for NSCLC, neratinib is in phase III of clinical development for the treatment of breast cancer, and afatinib is also in phase III development in head and neck cancer. Final results from clinical trials may lead to the potential approval of these agents in a variety of solid tumor malignancies.

Kinome-wide RNA interference screen reveals a role for PDK1 in acquired resistance to CDK4/6 inhibition in ER-positive breast cancer

Acquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) small-molecule inhibitors in breast cancer arises through mechanisms that are yet uncharacterized. In this study, we used a kinome-wide siRNA screen to identify kinases that, when downregulated, yield sensitivity to the CDK4/6 inhibitor ribociclib. In this manner, we identified 3-phosphoinositide-dependent protein kinase 1 (PDK1) as a key modifier of ribociclib sensitivity in estrogen receptor-positive MCF-7 breast cancer cells. Pharmacologic inhibition of PDK1 with GSK2334470 in combination with ribociclib or palbociclib, another CDK4/6 inhibitor, synergistically inhibited proliferation and increased apoptosis in a panel of ER-positive breast cancer cell lines. Ribociclib-resistant breast cancer cells selected by chronic drug exposure displayed a relative increase in the levels of PDK1 and activation of the AKT pathway. Analysis of these cells revealed that CDK4/6 inhibition failed to induce cell-cycle arrest or senescence. Mechanistic investigations showed that resistant cells coordinately upregulated expression of cyclins A, E, and D1, activated phospho-CDK2, and phospho-S477/T479 AKT. Treatment with GSK2334470 or the CDK2 inhibitor dinaciclib was sufficient to reverse these events and to restore the sensitivity of ribociclib-resistant cells to CDK4/6 inhibitors. Ribociclib, in combination with GSK2334470 or the PI3Kα inhibitor alpelisib, decreased xenograft tumor growth more potently than each drug alone. Taken together, our results highlight a role for the PI3K-PDK1 signaling pathway in mediating acquired resistance to CDK4/6 inhibitors. Cancer Res; 77(9); 2488-99. ©2017 AACR.