<div>Abstract<p>Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer with poor prognosis, and there is a critical need for novel therapeutic approaches. NEPC is associated with molecular perturbation of several pathways, including amplification of <i>MYCN</i>. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in the pathogenesis of neuroblastoma and other malignancies where it cooperates with N-Myc. We previously identified the first case of <i>ALK</i> F1174C-activating mutation in a patient with <i>de novo</i> NEPC who responded to the ALK inhibitor, alectinib. Here, we show that coactivation of ALK and N-Myc (ALK F1174C/N-Myc) is sufficient to transform mouse prostate basal stem cells into aggressive prostate cancer with neuroendocrine differentiation in a tissue recombination model. A novel gene signature from the ALK F1174C/N-Myc tumors was associated with poor outcome in multiple human prostate cancer datasets. ALK F1174C and ALK F1174C/N-Myc tumors displayed activation of the Wnt/β-catenin signaling pathway. Chemical and genetic ALK inhibition suppressed Wnt/β-catenin signaling and tumor growth <i>in vitro</i> in NEPC and neuroblastoma cells. ALK inhibition cooperated with Wnt inhibition to suppress NEPC and neuroblastoma proliferation <i>in vitro</i> and tumor growth and metastasis <i>in vivo</i>. These findings point to a role for ALK signaling in NEPC and the potential of cotargeting the ALK and Wnt/β-catenin pathways in ALK-driven tumors. Activated ALK and N-Myc are well known drivers in neuroblastoma development, suggesting potential similarities and opportunities to elucidate mechanisms and therapeutic targets in NEPC and vice versa.</p>Significance:<p>These findings demonstrate that coactivation of ALK and N-Myc induces NEPC by stimulating the Wnt/β-catenin pathway, which can be targeted therapeutically.</p></div>
<p>Figure S1 ALK genomic alterations and mRNA expression in human cancers. Figure S2 Efficacy of ALK inhibitors in prostate cancer cell lines. Table S1 Summary of mutations and their percentages identified by ctDNA profiling.</p>
// Sahithi Pamarthy 1 , Mukesh K. Jaiswal 1 , Arpita Kulshreshtha 1 , Gajendra K. Katara 1 , Alice Gilman-Sachs 1 , Kenneth D. Beaman 1 1 Department of Microbiology and Immunology, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA Correspondence to: Kenneth D. Beaman, e-mail: kenneth.beaman@rosalindfranklin.edu Keywords: a2V-ATPase, notch signaling, triple negative breast cancer, autophagy, bafilomycin Received: May 06, 2015 Accepted: September 07, 2015 Published: September 18, 2015 ABSTRACT Triple Negative Breast Cancer (TNBC) is a subtype of breast cancer with poor prognosis for which no targeted therapies are currently available. Notch signaling has been implicated in breast cancer but the factors that control Notch in TNBC are unknown. Because the Vacuolar ATPase has been shown to be important in breast cancer invasiveness, we investigated the role of a2-subunit isoform of Vacuolar ATPase (a2V) in regulating Notch signaling in TNBC. Confocal microscopy revealed that among all the 'a' subunit isoforms, a2V was uniquely expressed on the plasma membrane of breast cancer cells. Both a2V and NOTCH1 were elevated in TNBC tumors tissues and cell lines. a2V knockdown by siRNA as well as V-ATPase inhibition by Bafilomycin A1 (Baf A1) in TNBC cell lines enhanced Notch signaling by increasing the expression of Notch1 intracellular Domain (N1ICD). V-ATPase inhibition blocked NICD degradation by disrupting autophagy and lysosomal acidification as demonstrated by accumulation of LC3B and diminished expression of LAMP1 respectively. Importantly, treatment with Baf A1 or anti-a2V, a novel-neutralizing antibody against a2V hindered cell migration of TNBC cells. Our findings indicate that a2V regulates Notch signaling through its role in endolysosomal acidification and emerges as a potential target for TNBC.
<div>Abstract<p>Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer with poor prognosis, and there is a critical need for novel therapeutic approaches. NEPC is associated with molecular perturbation of several pathways, including amplification of <i>MYCN</i>. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in the pathogenesis of neuroblastoma and other malignancies where it cooperates with N-Myc. We previously identified the first case of <i>ALK</i> F1174C-activating mutation in a patient with <i>de novo</i> NEPC who responded to the ALK inhibitor, alectinib. Here, we show that coactivation of ALK and N-Myc (ALK F1174C/N-Myc) is sufficient to transform mouse prostate basal stem cells into aggressive prostate cancer with neuroendocrine differentiation in a tissue recombination model. A novel gene signature from the ALK F1174C/N-Myc tumors was associated with poor outcome in multiple human prostate cancer datasets. ALK F1174C and ALK F1174C/N-Myc tumors displayed activation of the Wnt/β-catenin signaling pathway. Chemical and genetic ALK inhibition suppressed Wnt/β-catenin signaling and tumor growth <i>in vitro</i> in NEPC and neuroblastoma cells. ALK inhibition cooperated with Wnt inhibition to suppress NEPC and neuroblastoma proliferation <i>in vitro</i> and tumor growth and metastasis <i>in vivo</i>. These findings point to a role for ALK signaling in NEPC and the potential of cotargeting the ALK and Wnt/β-catenin pathways in ALK-driven tumors. Activated ALK and N-Myc are well known drivers in neuroblastoma development, suggesting potential similarities and opportunities to elucidate mechanisms and therapeutic targets in NEPC and vice versa.</p>Significance:<p>These findings demonstrate that coactivation of ALK and N-Myc induces NEPC by stimulating the Wnt/β-catenin pathway, which can be targeted therapeutically.</p></div>
Abstract The development of resistance to cisplatin drugs significantly hinders successful ovarian cancer treatment. An emerging centralized mechanism of chemo-resistance is a dysregulation in tumor cell pH (alkaline intracellular, acidic extracellular). Vacuolar-ATPase (V-ATPase) is a key proton pump that alters the pH and mediates metastasis/chemo-resistance in tumor cells. The major pH sensing unit of the V-ATPase complex is the ‘a2’ isoform(V0a2) and has been previously shown to promote metastasis in ovarian cancer cells. Here, we show that V0a2 is over-expressed in acquired cisplatin resistant ovarian tumor cells (cis-A2780 and cis-TOV112D) at both mRNA and protein levels. The shRNA mediated inhibition of V0a2 activity in cisplatin resistant cells (sh-V0a2-cis) sensitized the cells to cisplatin with a 3 fold reduction in inhibitory concentration 50% [IC50] (sh-V0a2-cis, IC50 = 11.47 ± 3.02 μM, p<0.05) compared to control resistant cells [IC50 = 37.75 ± 6.62 μM] as determined by Alamar blue cell cytotoxicity assay. Suppression of V0a2 activity strongly reduced the cytosolic pH in the resistant tumor cells (cis-A2780- pH = 7.19 ± 0.03; sh-V0a2-cis- pH = 6.87 ± 0.04; cisplatin sensitive cells- pH = 6.96± 0.01). This cytosolic acidification significantly enhanced DNA damage by cisplatin-DNA adduct formation as revealed by FACS and immuno-fluorescence analysis (p<0.05). To further define the cisplatin mediated multi-factorial responses in resistant cells upon V0a2 inhibition, we performed a PCR array for cell death pathway related genes. Several pro-apoptotic genes including caspase 3, caspase 9, Apaf-1, FASL, CD40,CD40L, TNF, TNFR1 were up-regulated (p<0.05). Notably, caspase-1α was significantly up-regulated upon cisplatin treatment in sh-V0a2-cis compared to control resistant cells (p<0.05). Caspase-1α is known to induce apoptosis of neurons and macrophages and is recently indicated to be pro-apoptotic in ovarian cancer cells. Additionally, several autophagy related genes such as LC3A, IGFR, ESR1, IRGM were also found up-regulated upon V0a2 inhibition. Impaired autophagy in sh-V0a2-cis was further confirmed at protein level by FACS analysis using autophagy detection kit. Taken together, our findings suggest that the isoform specific inhibition of V-ATPase-V0a2 inhibits autophagy and activates caspase-1α upon cisplatin treatment in resistant ovarian cancer cells. V0a2 inhibition can therefore serve as a therapeutic strategy to overcome cisplatin resistance and in improving the treatment efficacy in chemo-resistant ovarian carcinoma. Citation Format: Arpita Kulshrestha, Gajendra K. Katara, Sahithi Pamarthy, Alice Gilman-Sachs, Kenneth D. Beaman. Reversal of cisplatin resistance in human ovarian cancer through autophagy inhibition and caspase 1-α activation: Role of tumor associated Vacuolar-ATPase. [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 4682.
Abstract Notch signaling plays an important role in regulation of innate immune responses and trophoblast function during pregnancy. To identify the role of Notch signaling in preterm labor, Notch receptors (Notch1-4), its ligands (DLL (Delta-like protein)-1/3/4), Jagged 1/2) and Notch-induced transcription factor Hes1 were assessed during preterm labor. Preterm labor was initiated on gestation day 14.5 by intrauterine (IU) injection of peptidoglycan (PGN) and polyinosinic:cytidylic acid (poly(I:C). Notch1, Notch2, Notch4, DLL-1 and nuclear localization of Hes1 were significantly elevated in uterus and placenta during PGN+poly(I:C)-induced preterm labor. Ex vivo , Gamma secretase inhibitor (GSI) (inhibitor of Notch receptor processing) significantly diminished the PGN+poly(I:C)-induced secretion of M1- and M2-associated cytokines in decidual macrophages and of proinflammatory cytokines (IFN-γ, TNF-α and IL-6) and chemokines (MIP-1β) in decidual and placental cells. Conversely, angiogenesis factors including Notch ligands Jagged 1/2 and DLL-4 and VEGF were significantly reduced in uterus and placenta during PGN+poly(I:C)-induced preterm labor. In vivo GSI treatment prevents PGN+poly(I:C)-induced preterm delivery by 55.5% and increased the number of live fetuses in-utero significantly compared to respective controls 48 hrs after injections. In summary, Notch signaling is activated during PGN+poly(I:C)-induced preterm labor, resulting in upregulation of pro-inflammatory responses and its inhibition improves in-utero survival of live fetuses.
The Vacuolar ATPase (V-ATPase) is a proton pump responsible for controlling the intracellular and extracellular pH of cells. The structure of V-ATPase has been highly conserved among all eukaryotic cells and is involved in diverse functions across species. V-ATPase is best known for its acidification of endosomes and lysosomes and is also important for luminal acidification of specialized cells. Several reports have suggested the involvement of V-ATPase in maintaining an alkaline intracellular and acidic extracellular pH thereby aiding in proliferation and metastasis of cancer cells respectively. Increased expression of V-ATPase and relocation to the plasma membrane aids in cancer modulates key tumorigenic cell processes like autophagy, Warburg effect, immunomoduation, drug resistance and most importantly cancer cell signaling. In this review, we discuss the direct role of V-ATPase in acidification and indirect regulation of signaling pathways, particularly Notch Signaling.
Abstract Ataxia Telangiectasia and Rad3-related (ATR) and its downstream effector Checkpoint Kinase 1 (CHK1) are central to the protection of stalled replication forks.Specific targeting of the ATR is synthetically lethal with multiple cancer-associated changes including oncogenic stress and defects in the DDR pathway and represents an emerging strategy to treat a broad spectrum of cancers. Atrin Pharmaceuticals has rationally designed a novel series of conformationally constrained macrocyclic ATR inhibitors with higher potency and selectivity than other ATR inhibitors currently in clinical development. An in-depth characterization of the ATRN series identified ATRN-119 as our lead compound with an in vitro enzyme IC50 of 20 nM and inhibition of ATR substrate CHK1 Ser345 phosphorylation in cells at an IC50 of 5 nM. ATRN-119 inhibits ATR in cells at much lower concentrations and demonstrates higher selectivity (>2000) for ATR over other closely-related PIK-kinases like ATM, DNA-PK and mTOR. Additionally, ATRN series have favorable ADME properties with increased water solubility and metabolic stability in human serum of up to 4 hours. Oral dosing of ATRN-119 showed significant antitumor effects in human pancreatic and colon cancer xenografts and in orthotopic ovarian Patient Derived Xenograft (PDX) tumors. Notably, hematological analysis of mice treated with daily oral dosing of ATRN-119 indicated no thrombocytopenia, anemia or neutropenia up to 4 weeks of treatment. Exploratory multiple high dosing toxicity studies in rats and dogs indicate significant exposure and good tolerability with lack of anemia or neutropenia. In a series of in-vitro cell viability assays, three dimensional organoid cultures and in-vivo combinationstudies, ATRN-119 showed significant synergism with various PARP inhibitors as well as restoration of PARPi sensitivity in PARPi resistant tumors. Our data suggests a new generation of highly potent and selective ATR inhibitors with favorable safety profile and a broad clinical therapeutic potential either as monotherapy, in combination with PARP inhibitors or as a synthetic lethal approach with key DDR mutations. Citation Format: Sahithi Pamarthy, Dansu Li, Ekaterine Goliadze, Tina Gill, Lanqi Jia, Erin George, Laura R. Butler, Ryan L. Ragland, Michel Afargan, Fiona A. Simpkins, Eric J. Brown, Oren Gilad. Highly specific macrocyclic ATR inhibitors for the targeted treatment of a broad spectrum of cancers showing lack of anemia or neutropenia in pre-clinical animal models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3498.
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