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Effective inhibition of angiogenesis targeting the tumor endothelial cells requires identification of key cellular and molecular mechanisms associated with survival of vasculatures within the tumor microenvironment. Intracellular autocrine (intracrine) VEGF production by endothelial cells plays a critical role on the vasculature homeostasis. In vitro breast cancer cell-stimulated activation of the unfolded protein response (UPR) of the endothelial cells contributes to maintenance of the intracrine VEGF levels in the endothelial cells through the upregulation of a previous undescribed downstream effector- αB-crystallin (CRYAB). siRNA-mediated knockdown of two major UPR proteins-inositol requiring kinase 1 and ATF6, led to attenuated CRYAB expression of the endothelial cells. Finally, inhibition of CRYAB blocked the breast cancer cell-stimulated increase in the endogenous VEGF levels of the endothelial cells. A VEGF limited proteolysis assay further revealed that CRYAB protected VEGF for proteolytic degradation. Here, we report that the molecular chaperone-CRYAB was significantly increased and colocalized with tumor vessels in a breast cancer xenograft. Specifically, neutralization of VEGF induced higher levels of CRYAB expression in the endothelial cells cocultured with MDA-MB-231 or the breast cancer xenograft with a significant survival benefit. However, knockdown of CRYAB had a greater inhibitory effect on endothelial survival. These findings underscore the importance of defining a role for intracrine VEGF signaling in sustaining aberrant tumor angiogenesis and strongly implicate UPR/CRYAB as dichotomous parts of a crucial regulation pathway for maintaining intracrine VEGF signaling.

Previous studies indicated that compounds termed Disulfide bond Disrupting Agents (DDAs) exhibit anti-cancer activity that is associated with downregulation of EGFR/HER1, HER2, and HER3, and activation of Death Receptors 4 and 5 (DR4/5). DDA-induced HER1-3 downregulation is preceded by disulfide-mediated oligomerization. In contrast, DDA-mediated DR4/5 oligomerization results in DR5 upregulation, and activation of DR4/5 pro-apoptotic signaling through Caspases 8 and 3. However, the precise mechanisms by which altered disulfide bonding stabilizes and activates DR5 are unknown. A recent report indicated that the extracellular domain of DR5 acts in an auto-inhibitory manner to prevent DR5 oligomerization and pro-apoptotic signaling in the absence of its ligand, TRAIL. A subsequent paper showed that the DR5 auto-inhibitory domain is a positive patch consisting of three basic residues. Importantly, the structure of the auto-inhibitory loop is formed by two disulfide bonds. We hypothesize that DDAs disrupt the disulfide bonds that make up the auto-inhibitory loop, resulting in DR5 oligomerization, and activation of Caspase 8/3-driven apoptosis in a TRAIL-independent manner. Due to their novel mechanisms of action, DDAs may overcome the pharmacological liabilities that have limited the efficacy of TRAIL analogs and DR5 agonist antibodies. Another unanswered question is how precisely DDAs alter DR5 and EGFR disulfide bonding. The direct targets of DDA action were revealed through affinity purification studies with biotinylated-DDA analogs. These studies identified the protein disulfide isomerases AGR2, ERp44, and PDIA1 as DDA target proteins, explaining how DDAs alter DR5 and EGFR disulfide bonding patterns. Consistent with this interpretation, knockdown of AGR2 or ERp44, or expression of catalytically null AGR2 or ERp44 mutants, mimicked DDAs in inducing disulfide-mediated DR5 oligomerization and Caspase 8 activation. Together, these results demonstrate a fundamentally novel, ligand-independent mechanism for activation of DR5 through DDA-mediated inhibition of the PDIs AGR2, ERp44, and PDIA1. Significantly, DDAs are the first identified active site inhibitors of AGR2 and ERp44. Citation Format: Brian K. Law, Mary E. Law, Elham Yaaghubi, Amanda Ghilardi, Brad J. Davis, Renan Ferreira, Samantha Eggleston, Jade Nguyen, Grace Alexandrow, Jin Koh, Sixue Chen, Chi-Wu Chiang, Coy Heldermon, Peter Norgaard, Ronald K. Castallano, Zaafir M. Dulloo. Disulfide isomerases AGR2, ERp44, and PDIA1 maintain death receptor 5 in an auto-inhibited, monomeric form. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6148.

Breast cancer mortality remains unacceptably high, indicating a need for safer and more effective therapeutic agents. Disulfide bond Disrupting Agents (DDAs) were previously identified as a novel class of anticancer compounds that selectively kill cancers that overexpress the Epidermal Growth Factor Receptor (EGFR) or its family member HER2. DDAs kill EGFR+ and HER2+ cancer cells via the parallel downregulation of EGFR, HER2, and HER3 and activation/oligomerization of Death Receptors 4 and 5 (DR4/5). However, the mechanisms by which DDAs mediate these effects are unknown. Affinity purification analyses employing biotinylated-DDAs reveal that the Protein Disulfide Isomerase (PDI) family members AGR2, PDIA1, and ERp44 are DDA target proteins. Further analyses demonstrate that shRNA-mediated knockdown of AGR2 and ERp44, or expression of ERp44 mutants, enhance basal and DDA-induced DR5 oligomerization. DDA treatment of breast cancer cells disrupts PDIA1 and ERp44 mixed disulfide bonds with their client proteins. Together, the results herein reveal DDAs as the first small molecule, active site inhibitors of AGR2 and ERp44, and demonstrate roles for AGR2 and ERp44 in regulating the activity, stability, and localization of DR4 and DR5, and activation of Caspase 8.

Disulfide bond-disrupting agents (DDAs) are a new chemical class of agents recently shown to have activity against breast tumors in animal models. Blockade of tumor growth is associated with downregulation of EGFR, HER2, and HER3 and reduced Akt phosphorylation, as well as the induction of endoplasmic reticulum stress. However, it is not known how DDAs trigger cancer cell death without affecting nontransformed cells. As demonstrated here, DDAs are the first compounds identified that upregulate the TRAIL receptor DR5 through transcriptional and post-transcriptional mechanisms to activate the extrinsic cell death pathway. At the protein level, DDAs alter DR5 disulfide bonding to increase steady-state DR5 levels and oligomerization, leading to downstream caspase 8 and 3 activation. DDAs and TRAIL synergize to kill cancer cells and are cytotoxic to HER2+ cancer cells with acquired resistance to the EGFR/HER2 tyrosine kinase inhibitor Lapatinib. Investigation of the mechanisms responsible for DDA selectivity for cancer cells reveals that DDA-induced upregulation of DR5 is enhanced in the context of EGFR overexpression. DDA-induced cytotoxicity is strongly amplified by MYC overexpression. This is consistent with the known potentiation of TRAIL-mediated cell death by MYC. Together, the results demonstrate selective DDA lethality against oncogene-transformed cells, DDA-mediated DR5 upregulation, and protein stabilization, and that DDAs have activity against drug-resistant cancer cells. Our results indicate that DDAs are unique in causing DR5 accumulation and oligomerization and inducing downstream caspase activation and cancer cell death through mechanisms involving altered DR5 disulfide bonding. DDAs thus represent a new therapeutic approach to cancer therapy.

Purpose: In acute myeloid leukemia (AML), recurrent DNA methyltransferase 3A (DNMT3A) mutations are associated with chemoresistance and poor prognosis, especially in advanced-age patients. Gene-expression studies in DNMT3A-mutated cells identified signatures implicated in deregulated DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here, we tested whether pharmacologically induced replication fork stalling, such as with cytarabine, creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. Experimental Design: Leukemia cell lines, genetic mouse models, and isogenic cells with and without DNMT3A(mut) were used to evaluate sensitivity to nucleoside analogues such as cytarabine in vitro and in vivo, followed by analysis of DNA damage and signaling, replication restart, and cell-cycle progression on treatment and after drug removal. Transcriptome profiling identified pathways deregulated by DNMT3A(mut) expression. Results: We found increased sensitivity to pharmacologically induced replication stress in cells expressing DNMT3A(R882)-mutant, with persistent intra–S-phase checkpoint activation, impaired PARP1 recruitment, and elevated DNA damage, which was incompletely resolved after drug removal and carried through mitosis. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine washout demonstrated a higher rate of fork collapse in DNMT3A(mut)-expressing cells. RNA-seq studies supported deregulated cell-cycle progression and p53 activation, along with splicing, ribosome biogenesis, and metabolism. Conclusions: Together, our studies show that DNMT3A mutations underlie a defect in recovery from replication fork arrest with subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during replication stress. See related commentary by Viny, p. 573

SUMMARYThe treatment of colorectal cancer (CRC) with FOLFOX shows some efficacy, but these tumors quickly develop resistance to this treatment. We have observed an increased phosphorylation of AKT1/mTOR/4EBP1 and levels of p21 in FOLFOX-resistant CRC cells. We have identified a small molecule, NSC49L, that stimulates protein phosphatase 2A (PP2A) activity, downregulates the AKT1/mTOR/4EBP1-axis, and inhibits p21 translation. We have provided evidence that NSC49L- and TRAIL-mediated sensitization is synergistically induced in p21-knockdown CRC cells, which is reversed in p21-overexpressing cells. p21 binds with procaspase 3 and prevents activation of caspase 3. We have shown that TRAIL induces apoptosis through the activation of caspase 3 by NSC49L-mediated downregulation of p21 translation, and thereby cleavage of procaspase 3 into caspase 3. NSC49L does not affect global protein synthesis. These studies provide a mechanistic understanding of NSC49L as a PP2A agonist, and how its combination with TRAIL sensitizes FOLFOX-resistant CRC cells.Graphical Abstract

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are recurrent in de novo acute myeloid leukemia (AML) and are associated with resistance to standard chemotherapy, disease relapse, and poor prognosis, especially in advanced-age patients. Previous gene expression studies in cells with DNMT3A mutations identified deregulation of cell cycle-related signatures implicated in DNA damage response and replication fork integrity, suggesting sensitivity to replication stress. Here we tested whether pharmacologically-induced replication fork stalling creates a therapeutic vulnerability in cells with DNMT3A(R882) mutations. We observed increased sensitivity to nucleoside analogs such as cytarabine in multiple cellular systems expressing mutant DNMT3A, ectopically or endogenously, in vitro and in vivo. Analysis of DNA damage signaling in response to cytarabine revealed persistent intra-S phase checkpoint activation, accompanied by accumulation of DNA damage in the DNMT3A(R882) overexpressing cells, which was only partially resolved after drug removal and carried through mitosis, resulting in micronucleation. Pulse-chase double-labeling experiments with EdU and BrdU after cytarabine wash-out demonstrated that cells with DNMT3A(mut) were able to restart replication but showed a higher rate of fork collapse. Gene expression profiling by RNA-seq identified deregulation of pathways associated with cell cycle progression and p53 activation, as well as metabolism and chromatin. Together, our studies show that cells with DNMT3A mutations have a defect in recovery from replication fork arrest and subsequent accumulation of unresolved DNA damage, which may have therapeutic tractability. These results demonstrate that, in addition to its role in epigenetic control, DNMT3A contributes to preserving genome integrity during DNA replication.

While HER2 and EGFR are overexpressed in breast cancers and multiple other types of tumors, the use of EGFR and/or HER2 inhibitors have failed to cure many cancer patients, largely because cancers acquire resistance to HER2/EGFR-specific drugs. Cancers that overexpress the HER-family proteins EGFR, HER2, and HER3 are uniquely sensitive to agents that disrupt HER2 and EGFR protein folding. We previously showed that disruption of disulfide bond formation by Disulfide Disrupting Agents (DDAs) kills HER2/EGFR overexpressing cells through multiple mechanisms. Herein, we show that interference with proline isomerization in HER2/EGFR overexpressing cells also induces cancer cell death. The peptidyl-prolyl isomerase inhibitor Cyclosporine A (CsA) selectively kills EGFR+ or HER2+ breast cancer cells in vitro by activating caspase-dependent apoptotic pathways. Further, CsA synergizes with the DDA tcyDTDO to kill HER2/EGFR overexpressing cells in vitro and the two agents cooperate to kill HER2+ tumors in vivo. There is a critical need for novel strategies to target HER2+ and EGFR+ cancers that are resistant to currently available mechanism-based agents. Drugs that target HER2/EGFR protein folding, including DDAs and CsA, have the potential to kill cancers that overexpress EGFR or HER2 through the induction of proteostatic synthetic lethality.

Despite new agent development and short-term benefits in patients with colorectal cancer (CRC), metastatic CRC cure rates have not improved due to high rates of 5-fluorouracil (5-FU)/leucovorin/oxaliplatin (FOLFOX)-resistance and a clinical therapeutic plateau. At the same time, this treatment regime leads to significant toxicity, cost, and patient inconvenience. Drug-resistance is linked to CRC stem cells, which are associated with the epidermal-to-mesenchymal transition (EMT) pathway. Thus, to optimally treat CRC, a therapy that can target the cell survival and EMT pathways in both CRC bulk and stem cell populations is critical. We recently identified a novel small molecule NSC30049 (7a) that is effective alone, and in combination potentiates 5-FU-mediated growth inhibition of CRC bulk, FOLFOX-resistant, and CRC stem cells both in vitro and in vivo models. In the present study, we report the synthesis and anti-CRC evaluation of several stable and effective 7a analogs. ASR352 (7b) was identified as one of the equipotent 7a analogs that inhibited the growth of CRC bulk cells, sensitized FOLFOX-resistant cells, and reduced the sphere formation capacity of CRC stem cells. It appears that the complex mechanism of cytotoxicity for 7b includes abrogation of 5-FU-induced the S phase, reduction of the phosphorylation of Chk1 at S317P, S345P and S296P, increased γH2AX staining, activation of caspase 3/PARP1 cleavage, and enhancement of Bax/Bcl2 ratio. Further 7b-mediated reduced phosphorylation of Chk1 was an indirect effect, since it did not inhibit Chk1 activity in an in vitro kinase assay. Our findings suggest that 7b as a single agent, or in combination with 5-FU can be developed as a therapeutic agent in CRC bulk, FOLFOX-resistant, and CRC stem cell populations for unmanageable metastatic CRC conditions., Graphical abstract Image 1, Highlights • Novel analogs of orphan molecule NSC30049 were synthesized. • ASR352 (7b) was identified as most cytotoxic to colorectal cancer (CRC) cells. • 7b inhibited the growth of CRC bulk and sensitized FOLFOX-resistant cells. • 7b reduced sphere formation capacity of CRC stem cells. • 7b abrogated 5-FU-induced the S phase and inhibited the phosphorylation of Chk1.

// Satya Narayan 1 , Aruna S. Jaiswal 2 , Ritika Sharma 1 , Akbar Nawab 1 , Lizette Vila Duckworth 3 , Brian K. Law 4 , Maria Zajac-Kaye 1 , Thomas J. George 2 , Jay Sharma 5 , Arun K. Sharma 6 and Robert A. Hromas 2 1 Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610, USA 2 Department of Medicine, University of Florida, Gainesville, FL 32610, USA 3 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32610, USA 4 Department of Pharmacology and Experimental Therapeutics, University of Florida, Gainesville, FL 32610, USA 5 Celprogen, Inc., Torrance, CA 90503, USA 6 Department of Pharmacology, Penn State Cancer Institute, Penn State College of Medicine, Hershey, PA 17033, USA Correspondence to: Satya Narayan, email: snarayan@ufl.edu Keywords: colorectal cancer, novel compound, FOLFOX-resistance, CRC stem cells, replication stress Received: April 28, 2017 Accepted: July 20, 2017 Published: August 01, 2017 ABSTRACT The 5-fluorouracil (5-FU) treatment induces DNA damage and stalling of DNA replication forks. These stalled replication forks then collapse to form one sided double-strand breaks, leading to apoptosis. However, colorectal cancer (CRC) stem cells rapidly repair the stalled/collapsed replication forks and overcome treatment effects. Recent evidence suggests a critical role of checkpoint kinase 1 (Chk1) in preventing the replicative stress. Therefore, Chk1 kinase has been a target for developing mono or combination therapeutic agents. In the present study, we have identified a novel orphan molecule NSC30049 (NSC49L) that is effective alone, and in combination potentiates 5-FU-mediated growth inhibition of CRC heterogeneous bulk and FOLFOX-resistant cell lines in culture with minimal effect on normal colonic epithelial cells. It also inhibits the sphere forming activity of CRC stem cells, and decreases the expression levels of mRNAs of CRC stem cell marker genes. Results showed that NSC49L induces 5-FU-mediated S-phase cell cycle arrest due to increased load of DNA damage and increased γ-H2AX staining as a mechanism of cytotoxicity. The pharmacokinetic analysis showed a higher bioavailability of this compound, however, with a short plasma half-life. The drug is highly tolerated by animals with no pathological aberrations. Furthermore, NSC49L showed very potent activity in a HDTX model of CRC stem cell tumors either alone or in combination with 5-FU. Thus, NSC49L as a single agent or combined with 5-FU can be developed as a therapeutic agent by targeting the Chk1 pathway in 5-FU-resistant CRC heterogeneous bulk and CRC stem cell populations.

Disulfide bond Disrupting Agents (DDAs) are a new chemical class of agents recently shown to have activity against breast tumors in animal models. However, it is unknown how DDAs trigger cancer cell death without affecting nontransformed cells. As demonstrated here, DDAs are the first compounds identified that upregulate the TRAIL receptor DR5 through both transcriptional and posttranscriptional mechanisms. At the protein level, DDAs alter DR5 disulfide bonding to increase steady-state DR5 levels and oligomerization, leading to downstream Caspase 8 and 3 activation. DDAs and TRAIL synergize to kill cancer cells and are cytotoxic to HER2+ cancer cells with acquired resistance to the EGFR/HER2 tyrosine kinase inhibitor. Investigation of the mechanisms responsible for DDA selectivity for cancer cells reveals that DDA-induced upregulation of DR5 is enhanced in the context of EGFR overexpression, and DDA-induced cytotoxicity is strongly amplified by MYC overexpression. Together, the results demonstrate selective DDA lethality against oncogene-transformed cells, DDA-mediated DR5 upregulation and protein stabilization, and DDAs against drug-resistant and metastatic cancer cells. DDAs thus represent a new therapeutic approach to cancer therapy.

Receptor tyrosine kinases (RTKs) have become major targets for anticancer therapy. However, resistance and signaling pathway redundancy has been problematic. The marine-derived apratoxins act complementary to direct kinase inhibitors by downregulating the levels of multiple of these receptors and additionally prevent the secretion of growth factors that act on these receptors by targeting Sec61α, therefore interfering with cotranslational translocation. We have profiled the synthetic, natural product-inspired apratoxin S4 against panels of cancer cells characterized by differential sensitivity to RTK inhibitors due to receptor mutations, oncogenic KRAS mutations, or activation of compensatory pathways. Apratoxin S4 was active at low-nanomolar to sub-nanomolar concentrations against panels of lung, head and neck, bladder, and pancreatic cancer cells, concomitant with the downregulation of levels of several RTKs, including EGFR, MET and others. However, the requisite concentration to inhibit certain receptors varied, suggesting some differential substrate selectivity in cellular settings. This selectivity was most pronounced in breast cancer cells, where apratoxin S4 selectively targeted HER3 over HER2 and showed greater activity against ER+ and triple negative breast cancer cells than HER2+ cancer cells. Depending on the breast cancer subtype, apratoxin S4 differentially downregulated transmembrane protein CDCP1, which is linked to metastasis and invasion in breast cancer and modulates EGFR activity. We followed the fate of CDCP1 through proteomics and found that nonglycosylated CDCP1 associates with chaperone HSP70 and HUWE1 that functions as an E3 ubiquitin ligase and presumably targets CDCP1, as well as potentially other substrates inhibited by apratoxins, for proteasomal degradation. By preventing cotranslational translocation of VEGF and other proangiogenic factors as well as VEGFR2 and other receptors, apratoxins also possess antiangiogenic activity, which was validated in endothelial cells where downregulation of VEGFR2 was observed, extending the therapeutic scope to angiogenic diseases., Graphical abstract Image 1, Highlights • Apratoxin S4 is a potent Sec61 inhibitor, , downregulating receptor tyrosine kinases (RTKs) and inhibiting proliferation and angiogenesis. • Inhibition of cotranslational translocation of RTKs due to Sec61 targeting was dependent on substrate and cellular context. • Most pronounced differential responses were observed in breast cancer subtypes, indicating preference for HER3 over HER2. • Proteasomal degradation of non-translocated membrane protein CDCP1 is suggested to be mediated by ubiquitin E3 ligase HUWE1. • Cell type dependent differential substrate selectivity might be explored for drug development and rational combination therapy.

EGFR, HER2, and HER3 contribute to the initiation and progression of human cancers, and are therapeutic targets for monoclonal antibodies and tyrosine kinase inhibitors. An important source of resistance to these agents arises from functional redundancy among EGFR, HER2, and HER3. EGFR family members contain conserved extracellular structures that are stabilized by disulfide bonds. Compounds that disrupt extracellular disulfide bonds could inactivate EGFR, HER2, and HER3 in unison. Here we describe the identification of compounds that kill breast cancer cells that overexpress EGFR or HER2. Cell death parallels downregulation of EGFR, HER2, and HER3. These compounds disrupt disulfide bonds and are termed Disulfide Bond Disrupting Agents (DDAs). DDA RBF3 exhibits anticancer efficacy in vivo at 40 mg/kg without evidence of toxicity. DDAs may complement existing EGFR-, HER2-, and HER3-targeted agents that function through alternate mechanisms of action, and combination regimens with these existing drugs may overcome therapeutic resistance.

Autophagy has been implicated in the progression and chemoresistance of various cancers. In this study, we have shown that osteosarcoma Saos-2 cells lacking ATG4B, a cysteine proteinase that activates LC3B, are defective in autophagy and fail to form tumors in mouse models. By combining in silico docking with in vitro and cell-based assays, we identified small compounds that suppressed starvation-induced protein degradation, LC3B lipidation, and formation of autophagic vacuoles. NSC185058 effectively inhibited ATG4B activity in vitro and in cells while having no effect on MTOR and PtdIns3K activities. In addition, this ATG4B antagonist had a negative impact on the development of Saos-2 osteosarcoma tumors in vivo. We concluded that tumor suppression was due to a reduction in ATG4B activity, since we found autophagy suppressed within treated tumors and the compound had no effects on oncogenic protein kinases. Our findings demonstrate that ATG4B is a suitable anti-autophagy target and a promising therapeutic target to treat osteosarcoma.

Acetyltransferase p300 (KAT3B) plays key roles in signaling cascades that support cancer cell survival and sustained proliferation. Thus, p300 represents a potential anticancer therapeutic target. To discover novel anticancer agents that target p300, we conducted a high-throughput screening campaign. A library of 622,079 compounds was assayed for cytotoxicity to the triple-negative breast cancer (TNBC) cell line MDA-MB-231 but not to the human mammary epithelial cells. The resulting compounds were tested in a biochemical assay for inhibiting the enzymatic activity of p300. One compound (L002, NSC764414) displayed an IC50 of 1.98 μmol/L against p300 in vitro, inhibited acetylation of histones and p53, and suppressed STAT3 activation in cell-based assays. L002 could be docked to the active site of the p300 catalytic domain. Biochemical tests of a series of related compounds revealed functional groups that may impact inhibitory potency of L002 against p300. Interestingly, these analogs showed inhibitory activities against the cellular paralog of p300 (CBP), p300/CBP-associated factor, and GCN5, but not to other acetyltransferases (KAT5, KAT6B, and KAT7), histone deacetylases, and histone methyltransferases. Among the NCI-60 panel of cancer cell lines, leukemia and lymphoma cell lines were extremely sensitive to L002, whereas it is toxic to only a limited number of cell lines derived from solid tumors. Notably, breast cancer cell lines, especially those derived from TNBC, were highly susceptible to L002. In vivo, it potently suppressed tumor growth and histone acetylation of MDA-MB-468 xenografts. Thus, these new acetyltransferase inhibitors are potential anticancer therapeutics. Mol Cancer Ther; 12(5); 610–20. ©2013 AACR.

Preclinical studies have established tumor angiogenesis as a potential therapeutic target for breast cancer. However, there is an urgent need to either improve existing anti-angiogenic agents or devise new anti-angiogenic therapy for a subset of breast cancer patients with resistance to current anti-angiogenic regimes. The purpose of this study is to develop an anti-angiogenic therapeutic model for breast cancer by a combination of 1) siRNA-based therapy intratumorally delivered by self-complementary adeno-associated virus serotype 2 (scAAV2) vector to target tumor vasculature, and 2) non-invasive monitoring for tumor response to anti-angiogenic agents by serial photoacoustic imaging. We have reported previously that under the stress conditions caused by tumor microenvironment or/and anti-VEGF therapies, endothelial cells adopt the up-regulation of IRE1α/XBP-1 and ATF6. This in turn maintains VEGF intracrine signaling for endothelial cell survival. Here we identified that scAAV2 septuplet-Y-F mutant vector was able to transfect mice microvascular endothelial cells with high efficiency. scAAV2 septuplet-tyrosine mutant vectors encoding the siRNAs against IRE1α or XBP-1 or ATF6 significantly inhibited breast cancer-induced angiogenesis in vitro by, in part, inhibiting endothelial cell survival. Acoustic-resolution photoacoustic microscopy (ARPAM) can provide non-invasive, label-free, high resolution vascular imaging. Utilizing ARAM, we showed that intratumoral delivering the siRNAs against IRE1α or XBP-1 or ATF6 by scAAV2 septuplet-tyrosine mutant vector resulted in a significant decrease in tumor growth and tumor angiogenesis in breast cancer xenograft models. These data have generated a proof-to-concept model with important implications for the development of novel anti-angiogenic targeted therapies for patients with breast cancer.

The epithelial to mesenchymal transition (EMT) is a process by which differentiated epithelial cells transition to a mesenchymal phenotype. EMT enables the escape of epithelial cells from the rigid structural constraints of the tissue architecture to a phenotype more amenable to cell migration and, therefore, invasion and metastasis. We characterized an in vivo model of EMT and discovered that marked changes in mitogenic signaling occurred during this process. DNA microarray analysis revealed that the expression of a number of genes varied significantly between post-EMT and pre-EMT breast cancer cells. Post-EMT cancer cells upregulated mRNA encoding c-Met and the PDGF and LPA receptors, and acquired increased responsiveness to HGF, PDGF, and LPA. This rendered the post-EMT cells responsive to the growth inhibitory effects of HGF, PDGF, and LPA receptor inhibitors/antagonists. Furthermore, post- EMT cells exhibited decreased basal Raf and Erk phosphorylation, and in comparison to pre-EMT cells, their proliferation was poorly inhibited by a MEK inhibitor. These studies suggest that therapies need to be designed to target both pre-EMT and post-EMT cancer cells and that signaling changes in post- EMT cells may allow them to take advantage of paracrine signaling from the stroma in vivo.

Carcinoma cells can acquire increased motility and invasiveness through epithelial-to-mesenchymal transition (EMT). However, the significance of EMT in cancer metastasis has been controversial, and the exact fates and functions of EMT cancer cells in vivo remain inadequately understood. Here, we tracked epithelial cancer cells that underwent inducible or spontaneous EMT in various tumor transplantation models. Unlike epithelial cells, the majority of EMT cancer cells were specifically located in the perivascular space and closely associated with blood vessels. EMT markedly activated multiple pericyte markers in carcinoma cells, in particular PDGFR-β and N-cadherin, which enabled EMT cells to be chemoattracted towards and physically interact with endothelium. In tumor xenografts generated from carcinoma cells that were prone to spontaneous EMT, a substantial fraction of the pericytes associated with tumor vasculature were derived from EMT cancer cells. Depletion of such EMT cells in transplanted tumors diminished pericyte coverage, impaired vascular integrity, and attenuated tumor growth. These findings suggest that EMT confers key pericyte attributes on cancer cells. The resulting EMT cells phenotypically and functionally resemble pericytes and are indispensable for vascular stabilization and sustained tumor growth. This study thus proposes a previously unrecognized role for EMT in cancer.

// Renan B. Ferreira 1, * , Mengxiong Wang 2, 3, * , Mary E. Law 2, 3 , Bradley J. Davis 2, 3 , Ashton N. Bartley 1 , Paul J. Higgins 4 , Michael S. Kilberg 5 , Katherine E. Santostefano 6 , Naohiro Terada 6 , Coy D. Heldermon 7 , Ronald K. Castellano 1 , Brian K. Law 2, 3 1 Department of Chemistry, University of Florida, Gainesville, FL 32611, USA 2 Department of Pharmacology & Therapeutics, University of Florida, Gainesville, FL 32610, USA 3 UF-Health Cancer Center, University of Florida, Gainesville, FL 32610, USA 4 Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY 12208, USA 5 Department of Biochemistry, University of Florida, Gainesville, FL, 32610, USA 6 Department of Pathology, Immunology, and Laboratory Medicine, Center for Cellular Reprogramming, University of Florida College of Medicine, Gainesville, FL 32610, USA 7 Department of Medicine, University of Florida, Gainesville, FL, 32610, USA * These authors have contributed equally to this work Correspondence to: Brian K. Law, email: bklaw@ufl.edu Ronald K. Castellano, email: castellano@chem.ufl.edu Keywords: HER2, EGFR, UPR, Akt, breast cancer Received: August 15, 2016 Accepted: February 12, 2017 Published: March 07, 2017 ABSTRACT Many breast cancer deaths result from tumors acquiring resistance to available therapies. Thus, new therapeutic agents are needed for targeting drug-resistant breast cancers. Drug-refractory breast cancers include HER2+ tumors that have acquired resistance to HER2-targeted antibodies and kinase inhibitors, and “Triple-Negative” Breast Cancers (TNBCs) that lack the therapeutic targets Estrogen Receptor, Progesterone Receptor, and HER2. A significant fraction of TNBCs overexpress the HER2 family member Epidermal Growth Factor Receptor (EGFR). Thus agents that selectively kill EGFR+ and HER2+ tumors would provide new options for breast cancer therapy. We previously identified a class of compounds we termed Disulfide bond Disrupting Agents (DDAs) that selectively kill EGFR+ and HER2+ breast cancer cells in vitro and blocked the growth of HER2+ breast tumors in an animal model. DDA-dependent cytotoxicity was found to correlate with downregulation of HER1-3 and Akt dephosphorylation. Here we demonstrate that DDAs activate the Unfolded Protein Response (UPR) and that this plays a role in their ability to kill EGFR+ and HER2+ cancer cells. The use of breast cancer cell lines ectopically expressing EGFR or HER2 and pharmacological probes of UPR revealed all three DDA responses: HER1-3 downregulation, Akt dephosphorylation, and UPR activation, contribute to DDA-mediated cytotoxicity. Significantly, EGFR overexpression potentiates each of these responses. Combination studies with DDAs suggest that they may be complementary with EGFR/HER2-specific receptor tyrosine kinase inhibitors and mTORC1 inhibitors to overcome drug resistance.

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER - adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) - promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents.

Aggressive cancers often express E-cadherin in cytoplasmic vesicles rather than on the plasma membrane and this may contribute to the invasive phenotype of these tumors. Therapeutic strategies are not currently available that restore the anti-invasive function of E-cadherin in cancers. MDA-MB-231 cells are a frequently used model of invasive triple-negative breast cancer, and these cells express low levels of E-cadherin that is mislocalized to cytoplasmic vesicles. MDA-MB-231 cell lines stably expressing wild-type E-cadherin or E-cadherin fused to glutathione S-transferase or green fluorescent protein were used as experimental systems to probe the mechanisms responsible for cytoplasmic E-cadherin localization in invasive cancers. Although E-cadherin expression partly reduced cell invasion in vitro, E-cadherin was largely localized to the cytoplasm and did not block the invasiveness of the corresponding orthotopic xenograft tumors. Further studies indicated that the glucocorticoid dexamethasone and the highly potent class I histone deacetylase (HDAC) inhibitor largazole cooperated to induce E-cadherin localization to the plasma membrane in triple-negative breast cancers, and to suppress cellular invasion in vitro. Dexamethasone blocked the production of the cleaved form of the CDCP1 (that is, CUB domain-containing protein 1) protein (cCDCP1) previously implicated in the pro-invasive activities of CDCP1 by upregulating the serine protease inhibitor plasminogen activator inhibitor-1. E-cadherin preferentially associated with cCDCP1 compared with the full-length form. In contrast, largazole did not influence CDCP1 cleavage, but increased the association of E-cadherin with γ-catenin. This effect on E-cadherin/γ-catenin complexes was shared with the nonisoform selective HDAC inhibitors trichostatin A (TSA) and vorinostat (suberoylanilide hydroxamic acid, SAHA), although largazole upregulated endogenous E-cadherin levels more strongly than TSA. These results demonstrate that glucocorticoids and HDAC inhibitors, both of which are currently in clinical use, cooperate to suppress the invasiveness of breast cancer cells through novel, complementary mechanisms that converge on E-cadherin.

Effective inhibition of angiogenesis targeting the tumor endothelial cells requires identification of key cellular and molecular mechanisms associated with survival of vasculatures within the tumor microenvironment. Intracellular autocrine (intracrine) VEGF production by endothelial cells plays a critical role on the vasculature homeostasis. In vitro breast cancer cell–stimulated activation of the unfolded protein response (UPR) of the endothelial cells contributes to maintenance of the intracrine VEGF levels in the endothelial cells through the upregulation of a previous undescribed downstream effector- αB-crystallin (CRYAB). siRNA-mediated knockdown of two major UPR proteins—inositol requiring kinase 1 and ATF6, led to attenuated CRYAB expression of the endothelial cells. Finally, inhibition of CRYAB blocked the breast cancer cell–stimulated increase in the endogenous VEGF levels of the endothelial cells. A VEGF limited proteolysis assay further revealed that CRYAB protected VEGF for proteolytic degradation. Here, we report that the molecular chaperone-CRYAB was significantly increased and colocalized with tumor vessels in a breast cancer xenograft. Specifically, neutralization of VEGF induced higher levels of CRYAB expression in the endothelial cells cocultured with MDA-MB-231 or the breast cancer xenograft with a significant survival benefit. However, knockdown of CRYAB had a greater inhibitory effect on endothelial survival. These findings underscore the importance of defining a role for intracrine VEGF signaling in sustaining aberrant tumor angiogenesis and strongly implicate UPR/CRYAB as dichotomous parts of a crucial regulation pathway for maintaining intracrine VEGF signaling. Mol Cancer Res; 9(12); 1632–43. ©2011 AACR.

Histone deacetylases (HDACs) are validated targets for anticancer therapy as attested by the approval of suberoylanilide hydroxamic acid (SAHA) and romidepsin (FK228) for treating cutaneous T cell lymphoma. We recently described the bioassay-guided isolation, structure determination, synthesis, and target identification of largazole, a marine-derived antiproliferative natural product that is a prodrug that releases a potent HDAC inhibitor, largazole thiol. Here, we characterize the anticancer activity of largazole by using in vitro and in vivo cancer models. Screening against the National Cancer Institute's 60 cell lines revealed that largazole is particularly active against several colon cancer cell types. Consequently, we tested largazole, along with several synthetic analogs, for HDAC inhibition in human HCT116 colon cancer cells. Enzyme inhibition strongly correlated with the growth inhibitory effects, and differential activity of largazole analogs was rationalized by molecular docking to an HDAC1 homology model. Comparative genomewide transcript profiling revealed a close overlap of genes that are regulated by largazole, FK228, and SAHA. Several of these genes can be related to largazole's ability to induce cell cycle arrest and apoptosis. Stability studies suggested reasonable bioavailability of the active species, largazole thiol. We established that largazole inhibits HDACs in tumor tissue in vivo by using a human HCT116 xenograft mouse model. Largazole strongly stimulated histone hyperacetylation in the tumor, showed efficacy in inhibiting tumor growth, and induced apoptosis in the tumor. This effect probably is mediated by the modulation of levels of cell cycle regulators, antagonism of the AKT pathway through insulin receptor substrate 1 down-regulation, and reduction of epidermal growth factor receptor levels.

EGFR and/or HER2 are frequently overexpressed oncoproteins in HER2+ breast cancer, Triple Negative Breast Cancer (TNBC), and Inflammatory Breast Cancer (IBC). However, EGFR and/or HER2 targeting agents have failed to make major advances in improving survival for these cancer patients, largely because these cancers frequently develop resistance to these drugs. The identification of novel treatment strategies is critically needed. Overexpression of EGFR and/or HER2 presents a severe burden to the secretary pathway, as HER-family proteins EGFR, HER2, HER3 share conserved extracellular cysteine-rich and proline-rich repeats that form numerous disulfide bonds and control cis-trans isomerization in the protein folding processes. We demonstrated that the disulfide bond disrupting Agents (DDAs) selectively killed EGFR+ and/or HER2+ breast cancer cells in vitro and decreased tumor growth of HER2+ cancer in a xenograft mouse model. DDA-dependent anti-cancer actions resulted from downregulation of EGFR/HER2/HER3, induction of AKT dephosphorylation, and activation of Endoplasmic Reticulum (ER) stress. We also investigated a novel proteotoxic combination therapy employing DDAs and Proline Isomerase Inhibitors (PIIs) in treating breast cancers. PIIs potentiated the efficacy of DDAs in downregulating EGFR/HER2/HER3 levels and initiating ER stress, and the combination was highly synergistic in reducing EGFR+ and/or HER2+ breast cancer cell viability. Importantly, the combination of DDAs and PIIs overcame single drug resistance, significantly decreased tumor volumes and increased the survival of xenograft mice. Evidence that the drug combination was effective against metastatic breast cancer cells is also provided. Citation Format: Mengxiong Wang, Renan Ferreira, Mary Law, Elham Yaaghubi, Coy Heldermon, Ronald Castellano, Brian Law. A novel proteotoxic combination therapy for EGFR+ and HER2+ cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 447.

Epithelial-mesenchymal transition (EMT) has pivotal roles during embryonic development and carcinoma progression. Members of the Snai1 family of zinc finger transcription factors are central mediators of EMT and induce EMT in part by directly repressing epithelial markers such as E-cadherin, a gatekeeper of the epithelial phenotype and a suppressor of tumor invasion. However, the molecular mechanism underlying Snai1-mediated transcriptional repression remains incompletely understood. Here we show that Snai1 physically interacts with and recruits the histone demethylase LSD1 (KDM1A) to epithelial gene promoters. LSD1 removes dimethylation of lysine 4 on histone H3 (H3K4m2), a covalent histone modification associated with active chromatin. Importantly, LSD1 is essential for Snai1-mediated transcriptional repression and for maintenance of the silenced state of Snai1 target genes in invasive cancer cells. In the absence of LSD1, Snai1 fails to repress E-cadherin. In cancer cells in which E-cadherin is silenced, depletion of LSD1 results in partial de-repression of epithelial genes and elevated H3K4m2 levels at the E-cadherin promoter. These results underline the critical role of LSD1 in Snai1-dependent transcriptional repression of epithelial markers and suggest that the LSD1 complex could be a potential therapeutic target for prevention of EMT-associated tumor invasion.

NF-kappaB is activated in many types of cancer. Phosphorylation of p65 at serine 276 is required for the expression of a subset of NF-kappaB regulated genes, including vascular cell adhesion molecule-1 (VCAM-1) and interleukin-8 (IL-8). Thus, inhibition of serine 276 phosphorylation may prevent metastasis and angiogenesis in certain tumor types. Using in silico molecular docking, small molecules that are predicted to bind to a structural pocket near serine 276 were identified. One compound, NSC-127102, hinders serine 276 phosphorylation and the expression of IL-8 and VCAM-1. Small molecules such as NSC-127102 may be optimized for the future treatment of cancer.

DNA alkylation-induced damage is one of the most efficacious anti-cancer therapeutic strategies. Increased DNA alkylation and decreased DNA repair capacity in cancer cells is responsible for the effectiveness of DNA-alkylating therapies. 5’-Flap endonuclease 1 (Fen1) is an important enzyme involved in base excision repair (BER), specifically in long-patch (LP)-BER. Using the site-directed mutagenesis approach, we have identified an important role for amino acid Asp181 of Fen1 in its endonuclease activity. The Asp181 is thought be involved in Mg 2+ binding in the active site. Using structure-based molecular docking of Fen1 targeted to its metal-binding pocket M2 (Mg 2+ site), we have identified a potent small molecular weight inhibitor (SMI; NSC-281680) that efficiently blocks the LP-BER. In this study, we have demonstrated that the interaction of this SMI with Fen1 blocked its endonuclease activity, thereby blocking the LP-BER and enhancing the cytotoxic effect of DNAalkylating agent, Temozolomide (TMZ) in mismatch repair (MMR)-deficient and MMR-proficient colon cancer cells. The results further suggest that blockade of LP-BER by NSC-281680 may bypass other drug resistance mechanisms such as mismatch repair (MMR) defects. Therefore, our findings provide groundwork for the development of highly specific and safer structure-based small molecular inhibitors targeting the BER pathway, which can be used along with existing chemotherapeutic agents, like TMZ, as combination therapy for the treatment of colorectal cancer.

The basal-like subtype of breast cancer is associated with invasiveness, high rates of postsurgical recurrence, and poor prognosis. Aside from inactivation of the BRCA1 tumor-suppressor gene, little is known concerning the mechanisms that cause basal breast cancer or the mechanisms responsible for its invasiveness. Here, we show that the heterogeneous mouse mammary tumor virus-cyclin D1-Cdk2 (MMTV-D1K2) transgenic mouse mammary tumors contain regions of spindle-shaped cells expressing both luminal and myoepithelial markers. Cell lines cultured from these tumors exhibit the same luminal/myoepithelial mixed-lineage phenotype that is associated with human basal-like breast cancer and express a number of myoepithelial markers including cytokeratin 14, P-cadherin, α smooth muscle actin, and nestin. The MMTV-D1K2 tumor-derived cell lines form highly invasive tumors when injected into mouse mammary glands. Invasion is associated with E-cadherin localization to the cytoplasm or loss of E-cadherin expression. Cytoplasmic E-cadherin correlates with lack of colony formation in vitro and β-catenin and p120ctn localization to the cytoplasm. The data suggest that the invasiveness of these cell lines results from a combination of factors including mislocalization of E-cadherin, β-catenin, and p120ctn to the cytoplasm. Nestin expression and E-cadherin mislocalization were also observed in human basal-like breast cancer cell lines, suggesting that these results are relevant to human tumors. Together, these results suggest that abnormal Cdk2 activation may contribute to the formation of basal-like breast cancers.

Protein serine/threonine phosphatase 2A (PP2A) activity must be tightly controlled to maintain cell homeostasis. Here, we report the identification of a previously uncharacterized mammalian protein, type 2A-interacting protein (TIP), as a novel regulatory protein of PP2A and the PP2A-like enzymes PP4 and PP6. TIP is a ubiquitously expressed protein and parallels the distribution of the PP2A catalytic subunit. Unlike its role in yeast, TIP does not interact with the mammalian homolog of type 2A-associated protein of 42 kDa (Tap42), alpha4, but instead associates with PP2A, PP4 and PP6 catalytic subunits independently of mammalian target of rapamycin kinase activity. Interestingly, the 20 kDa TIP splice variant TIP_i2, which lacks amino acids 173-272 of TIP's C-terminus, does not interact with PP2A; this finding indicates that residues 173-272 are important for the assembly of the TIP.phosphatase complex. In contrast to purified PP2A holoenzymes, TIP.PP2A complexes are devoid of phosphatase activity. Furthermore, alterations in the cellular levels of TIP influence the phosphorylation state of a specific protein substrate of ataxia-telangiectasia mutated (ATM)/ATM- and Rad3-related (ATR) kinases. Elevated levels of TIP result in an increase in the phosphorylation state of this protein substrate, whereas TIP-depleted cells exhibit a significant decrease in this protein's phosphorylation state, which is reversed by treatment with the PP2A inhibitor okadaic acid. These results indicate TIP is a novel inhibitory regulator of PP2A and implicate a role for TIP.PP2A complexes within the ATM/ATR signaling pathway controlling DNA replication and repair.

Rapamycin and its derivatives are promising anticancer agents, but the exact mechanisms by which these drugs induce cell cycle arrest and inhibit tumor growth are unknown. A biochemical analysis of human mammary tumor cell lines indicated that rapamycin-induced antiproliferative effects correlated with down-regulation of cellular p21 levels and the levels of p21 in cyclin-dependent kinase (Cdk) 2 and 4 complexes. Cyclin D1 overexpression reversed rapamycin action and this reversal correlated with increased levels of cellular p21, higher levels of p21 associated with Cdk2, and stabilization of cyclin D1/Cdk2/p21/proliferating cell nuclear antigen (PCNA) complexes. Experiments using a novel cyclin D1-Cdk2 fusion protein or a kinase-dead mutant of the fusion protein indicated that reversal of rapamycin action required not only the formation of complexes with p21 and PCNA but also complex-associated kinase activity. Similar results were observed in vivo. The rapamycin derivative RAD001 (everolimus) inhibited the growth of mouse mammary tumors, which correlated with the disruption of cyclin D1/Cdk2 complexes. The potential implications of these results with respect to the use of rapamycin derivatives in breast cancer therapy are discussed. (Cancer Res 2006; 66(2): 1070-80)

Rapamycin and its derivatives are promising therapeutic agents with both immunosuppressant and anti-tumor properties. These rapamycin actions are mediated through the specific inhibition of the mTOR protein kinase. mTOR serves as part of an evolutionarily conserved signaling pathway that controls the cell cycle in response to changing nutrient levels. The mTOR signaling network contains a number of tumor suppressor genes including PTEN, LKB1, TSC1, and TSC2, and a number of proto-oncogenes including PI3K, Akt, and eIF4E, and mTOR signaling is constitutively activated in many tumor types. These observations point to mTOR as an ideal target for anti-cancer agents and suggest that rapamycin is such an agent. In fact, early preclinical and clinical studies indicate that rapamycin derivatives have efficacy as anti-tumor agents both alone, and when combined with other modes of therapy. Rapamycin appears to inhibit tumor growth by halting tumor cell proliferation, inducing tumor cell apoptosis, and suppressing tumor angiogenesis. Rapamycin immunosuppressant actions result from the inhibition of T and B cell proliferation through the same mechanisms that rapamycin blocks cancer cell proliferation. Therefore, one might think that rapamycin-induced immunosuppression would be detrimental to the use of rapamycin as an anti-cancer agent. To the contrary, rapamycin decreases the frequency of tumor formation that occurs in organ transplant experiments when combined with the widely used immunosuppressant cyclosporine compared with the tumor incidence observed when cyclosporine is used alone. The available evidence indicates that with respect to tumor growth, rapamycin anti-cancer activities are dominant over rapamycin immunosuppressant effects.

Transforming growth factor beta (TGF-beta) induces cell cycle arrest of most nontransformed epithelial cell lines. In contrast, many human carcinomas are refractory to the growth-inhibitory effect of TGF-beta. TGF-beta overexpression inhibits tumorigenesis, and abolition of TGF-beta signaling accelerates tumorigenesis, suggesting that TGF-beta acts as a tumor suppressor in mouse models of cancer. A screen to identify agents that potentiate TGF-beta-induced growth arrest demonstrated that the potential anticancer agent rapamycin cooperated with TGF-beta to induce growth arrest in multiple cell lines. Rapamycin also augmented the ability of TGF-beta to inhibit the proliferation of E2F1-, c-Myc-, and (V12)H-Ras-transformed cells, even though these cells were insensitive to TGF-beta-mediated growth arrest in the absence of rapamycin. Rapamycin potentiation of TGF-beta-induced growth arrest could not be explained by increases in TGF-beta receptor levels or rapamycin-induced dissociation of FKBP12 from the TGF-beta type I receptor. Significantly, TGF-beta and rapamycin cooperated to induce growth inhibition of human carcinoma cells that are resistant to TGF-beta-induced growth arrest, and arrest correlated with a suppression of Cdk2 kinase activity. Inhibition of Cdk2 activity was associated with increased binding of p21 and p27 to Cdk2 and decreased phosphorylation of Cdk2 on Thr(160). Increased p21 and p27 binding to Cdk2 was accompanied by decreased p130, p107, and E2F4 binding to Cdk2. Together, these results indicate that rapamycin and TGF-beta cooperate to inhibit the proliferation of nontransformed cells and cancer cells by acting in concert to inhibit Cdk2 activity.

ErbB2/Neu destabilizes the cyclin-dependent kinase (Cdk) inhibitor p27 and increases expression of cyclin D1. Therefore, we studied the roles of p27 and cyclin D1 in ErbB2-mediated mammary epithelial cell transformation. Overexpression of ErbB2 or cyclin D1 in p27(+/-) primary murine mammary epithelial cells resulted in increased proliferation, cyclin D1 nuclear localization, and colony formation in soft agar compared to those in p27(+/+) cells. In contrast, ErbB2- or cyclin D1-overexpressing p27(-/-) cells displayed reduced proliferation, anchorage-independent growth, Cdk4 activity, cyclin D1 expression, and cyclin D1 nuclear localization compared to wild-type cells. A cyclin D1 mutation in its nuclear export sequence (T286A) partially rescued nuclear localization of cyclin D1 in p27(-/-) cells but did not increase proliferation or Cdk4 kinase activity. Overexpression of E2F1, however, increased proliferation to the same degree in p27(+/+), p27(+/-), and p27(-/-) cells. Mammary glands from MMTV (mouse mammary tumor virus)-neu/p27(+/-) mice exhibited alveolar hyperplasia, enhanced proliferation, decreased apoptosis, and accelerated tumor formation compared to MMTV-neu/p27(+/+) glands. However, MMTV-neu/p27(-/-) glands showed decreased proliferation, cyclin D1 expression, and Cdk4 activity, as well as markedly prolonged tumor latency, compared to MMTV-neu/p27(+/+) glands. These results suggest that p27(+/-) mammary epithelium may be more susceptible to oncogene-induced tumorigenesis, whereas p27-null glands, due to severely impaired cyclin D1/Cdk4 function, are more resistant to transformation.

Unmet needs in the breast cancer field include the development of effective treatments for 1) drug-resistant HER2+ breast cancers, 2) Triple-Negative (HER2-, ER-, PR-) Breast Cancers (TNBCs), and 3) Inflammatory Breast Cancers (IBCs). Treatment-refractory HER2+ tumors retain HER2 expression after acquiring drug resistance, and large fractions of TNBCs and IBCs overexpress either HER2 or EGFR. Thus, drugs that selectively kill breast tumors that overexpress EGFR or HER2 could benefit patients suffering from these three classes of treatment-refractory tumors. Our team has identified a class of compounds termed Disulfide bond Disrupting Agents (DDAs) that selectively kill cancer cells that overexpress HER2 or EGFR. DDA-mediated cancer-selective cytotoxicity results from A) downregulation of the EGFR/HER2/HER3 proteins, B) inactivation of the Akt oncogene, and C) activation of the Unfolded Protein Response (UPR). Importantly DDAs only elicit these responses in cancer cells that overexpress EGFR or HER2. DDAs exhibit striking anticancer activity in human HER2+ xenograft tumors grown orthotopically in mice, with no evidence of toxicity. DDAs function through mechanisms distinct from those of HER2 specific monoclonal antibodies and tyrosine kinase inhibitors, and biochemical studies indicate that DDAs are complementary with Lapatinib with respect to blockade of HER2 driven proliferative and survival signaling. Current efforts are directed toward identifying mechanism-based drugs that synergize with DDAs to potentiate HER1-3 downregulation, Akt inhibition, and UPR, and to kill HER2+/EGFR+ patient-derived xenograft tumors that are refractory to current therapies. Citation Format: Brian K. Law, Mary E. Law, Renan B. Ferreira, Mengxiong Wang, Bradley J. Davis, Ronald K. Castellano, Coy D. Heldermon. Multifaceted targeting of drug-resistant EGFR+ and HER2+ breast tumors [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 4050. doi:10.1158/1538-7445.AM2017-4050

The TGF-beta signaling pathway controls multiple functions of cancer cells and the surrounding stromal tissue. Some TGF-beta actions suppress cancer formation, while others contribute to tumor progression. Evidence supporting a tumor suppressive role for the TGF-beta/Smad signaling axis is presented here. These data are compiled from cell culture studies, animal models, analyses of human tumors, and investigations of polymorphisms of TGF-beta pathway components and their associated cancer risk. Therapeutic strategies for cancer treatment involving either restoring or potentiating TGF-beta tumor suppressive activities, or blocking TGF-beta tumor promoting functions are considered.

Phosphorylation of the p65 subunit of NF-kappaB is required for its transcriptional activity. Recent reports show that phosphorylation of p65 at serine 276 regulates only a subset of genes, such as those encoding IL-6, IL-8, Gro-beta, and ICAM-1. In order to identify additional genes regulated by serine 276 phosphorylation, HepG2 hepatoma cells were infected with adenoviruses encoding either wild-type p65 or the S276A mutant of p65, followed by DNA microarray analysis. The results show that mutation of serine 276 affected the expression of several genes that encode proteins involved in cell cycle regulation, signal transduction, transcription, and metabolism. Notably, expression of S276A increased the mRNA and protein level of p27, a cell cycle inhibitory protein, which led to an increased association of p27 with cdk2, and inhibition of cdk2 activity. Furthermore, while wild-type NF-kappaB is known to increase cell proliferation in a number of different cancer cell lines, our data shows that S276A inhibited cell proliferation. Evidence is mounting that NF-kappaB plays a pivotal role in oncogenesis. Therapeutic agents that regulate the phosphorylation of serine 276 and p27 gene expression, therefore, may be useful as anti-cancer agents in the future.

Normal tissue homeostasis is maintained by strict regulation of interactions between cells and their microenvironment. How a cell responds to stimulatory and inhibitory signals it receives from the microenvironment will directly impact whether or not that particular cell will proceed through the cell cycle and proliferate or stop cell cycle progression and undergo assessment. When cells no longer respond to their microenvironmental cues and proliferate autonomously, tumors arise. The major known negative regulators of cell proliferation are the transforming growth factor βs (TGF-βs). The TGF-β signaling pathways are tumor suppressive, yet once tumors have developed, TGF-β signaling can enhance tumor progression.

Cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes are present at high frequency in human breast cancer cell lines, but the significance of this observation is unknown. This report shows that expression of a cyclin D1–Cdk2 fusion protein under the control of the mouse mammary tumor virus (MMTV) promoter results in mammary gland hyperplasia and fibrosis, and mammary tumors. Cell lines isolated from MMTV–cyclin D1–Cdk2 (MMTV-D1K2) tumors exhibit Rb and p130 hyperphosphorylation and up-regulation of the protein products of E2F-dependent genes. These results suggest that cyclin D1/Cdk2 complexes may mediate some of the transforming effects that result from cyclin D1 overexpression in human breast cancers. MMTV-D1K2 cancer cells express the hepatocyte growth factor (HGF) receptor, c-Met. MMTV-D1K2 cancer cells also secrete transforming growth factor β (TGFβ), but are relatively resistant to TGFβ antiproliferative effects. Fibroblasts derived from MMTV-D1K2 tumors secrete factors that stimulate the proliferation of MMTV-D1K2 cancer cells, stimulate c-Met tyrosine phosphorylation, and stimulate the phosphorylation of the downstream signaling intermediates p70s6k and Akt on activating sites. Together, these results suggest that deregulation of the Cdk/Rb/E2F axis reprograms mammary epithelial cells to initiate a paracrine loop with tumor-associated fibroblasts involving TGFβ and HGF, resulting in desmoplasia. The MMTV-D1K2 mice should provide a useful model system for the development of therapeutic approaches to block the stromal desmoplastic reaction that likely plays an important role in the progression of multiple types of human tumors. [Cancer Res 2007;67(7):3135–44]

Recently approved chemotherapeutic agents to treat colorectal cancer (CRC) have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β) activity. Temozolomide (TMZ), an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER) pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715). In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP) site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC50 of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.

Metastasis is a primary cause of mortality due to cancer. Early metastatic growth involves both a remodeling of the extracellular matrix surrounding tumors and invasion of tumors across the basement membrane. Up-regulation of extracellular matrix degrading proteases such as urokinase plasminogen activator (uPA) and matrix metalloproteinases has been reported to facilitate tumor cell invasion. Autocrine transforming growth factor-beta (TGF-beta) signaling may play an important role in cancer cell invasion and metastasis; however, the underlying mechanisms remain unclear. In the present study, we report that autocrine TGF-beta supports cancer cell invasion by maintaining uPA levels through protein secretion. Interestingly, treatment of paracrine/exogenous TGF-beta at higher concentrations than autocrine TGF-beta further enhanced uPA expression and cell invasion. The enhanced uPA expression by exogenous TGF-beta is a result of increased uPA mRNA expression due to RNA stabilization. We observed that both autocrine and paracrine TGF-beta-mediated regulation of uPA levels was lost upon depletion of Smad4 protein by RNA interference. Thus, through the Smad pathway, autocrine TGF-beta maintains uPA expression through facilitated protein secretion, thereby supporting tumor cell invasiveness, whereas exogenous TGF-beta further enhances uPA expression through mRNA stabilization leading to even greater invasiveness of the cancer cells.

The failure of standard of care treatment for patients diagnosed with glioblastoma (GBM) coupled with the highly vascularized nature of this solid tumor has led to the consideration of agents that target vascular endothelial growth factor (VEGF) or its receptors, as alternative therapeutic strategies for this disease. While early clinical studies determined that recurrent GBMs respond favorably to bevacizumab (BVZ), progression of tumors after anti-VEGF targeted therapy is inevitable and in many instances the disease presents with a more aggressive phenotype. Recently completed phase III trials evaluating BVZ in combination with chemoradiation in newly diagnosed GBM also failed to establish an overall survival benefit. Hence there is a critical need to understand why anti-angiogenic therapies are ineffective in GBM patients. In particular, knowledge of the mechanisms responsible for adaptive resistance to VEGF/VEGFR targeting agents, that characterize recurring tumors, will be needed if significant advancement in prolonging survival rates of GBM patients is to be achieved. The results reported herein, suggest a potential mechanism by which anti-VEGF/VEGFR therapies regulate the enhanced invasive phenotype through a pathway that involves transforming growth factor beta (TGFβ) receptor (TGFβR) and chemokine receptor CXCR4. The VEGFR signaling inhibitors (Cediranib and Vandetanib) elevated the expression of CXCR4 in VEGFR-expressing primary patient-derived GBM cell lines and tumors, and enhanced the in vitro migration of these lines toward CXCL12. The combination of Cediranib and the CXCR4 antagonist AMD3100/Plerixafor provided a greater survival benefit to tumor-bearing animals, compared to monotherapies with these agents. The upregulation of CXCR4 by VEGFR inhibitors was dependent on TGFβ/TGFβR, but not HGF/MET, signaling activity, suggesting a mechanism of crosstalk among VEGF/VEGFR, CXCL12/CXCR4, and TGFβ/TGFβR pathways in the malignant phenotype of recurrent tumors after anti-VEGF/VEGFR therapies. Thus, the combination of VEGFR, CXCR4, and TGFβR inhibitors could provide an alternative strategy to halt GBM progression.

Background The transforming growth factor-β (TGF-β) signaling pathway functions to prevent tumorigenesis, and loss of sensitivity to TGF-β-mediated cell cycle arrest is nearly ubiquitous among human cancers. Our previous studies demonstrated that rapamycin potentiates TGF-β-induced cell cycle arrest in nontransformed epithelial cells and partially restores TGF-β-induced growth arrest of some human cancer cell lines. Growth arrest correlated with increased binding of p21 and p27 to cyclin-dependent kinase-2 (Cdk2), and inhibition of Cdk2 kinase activity. However, it was unclear how TGF-β caused increased binding of p21 and p27 to Cdk2. Methods Cell fractionation and immunofluorescence microscopy experiments were performed to examine the effect of TGF-β on the intracellular localization of Cdk2, p21, and p27. Kinase assays were performed on cytoplasmic and nuclear extracts to determine how TGF-β altered Cdk2 activity in both subcellular compartments. Results In breast epithelial cells treatment with TGF-β induced a decrease in nuclear Cdk2 concentrations and relocalization of Cdk2 to the cytoplasm. Cdk2 relocalization to the cytoplasm correlated with dephosphorylation of nuclear retinoblastoma tumor suppressor protein and decreased nuclear Cdk2 activity. In these epithelial cell lines, p21 and p27 were localized primarily in the cytoplasm. Decreases in nuclear Cdk2 concentrations correlated with increased binding of Cdk2 to cytoplasmic p21 and p27. Conclusion Cooperative growth arrest induced by treatment with TGF-β + rapamycin causes inhibition of nuclear Cdk2 activity through multiple mechanisms, including Cdk2 relocalization to the cytoplasm, increased p27 and p21 binding to Cdk2, and increased phosphorylation of nuclear Cdk2 on its inhibitory site, Tyr15.

Transforming growth factor beta (TGF-beta) plays an essential role in a wide array of cellular processes. The most well studied TGF-beta response in normal epithelial cells is growth inhibition. In some cell types, TGF-beta induces an epithelial to mesenchymal transition (EMT). NMuMG is a nontransformed mouse mammary gland epithelial cell line that exhibits both a growth inhibitory response and an EMT response to TGF-beta, rendering NMuMG cells a good model system for studying these TGF-beta effects.A National Institutes of Aging mouse 15,000 cDNA microarray was used to profile the gene expression of NMuMG cells treated with TGF-beta1 for 1, 6, or 24 hours. Data analyses were performed using GenePixPro and GeneSpring software. Selected microarray results were verified by northern analyses.Of the 15,000 genes examined by microarray, 939 were upregulated or downregulated by TGF-beta. This represents approximately 10% of the genes examined, minus redundancy. Seven genes previously not known to be regulated by TGF-beta at the transcriptional level (Akt and RhoB) or not at all (IQGAP1, mCalpain, actinin alpha3, Ikki, PP2A-PR53), were identified and their regulation by TGF-beta verified by northern blotting. Cell cycle pathway examination demonstrated downregulation of cyclin D2, c-myc, Id2, p107, E2F5, cyclin A, cyclin B, and cyclin H. Examination of cell adhesion-related genes revealed upregulation of c-Jun, alpha-actinin, actin, myosin light chain, p120cas catenin (Catns), alpha-integrin, integrin beta5, fibronectin, IQGAP1, and mCalpain.Using a cDNA microarray to examine TGF-beta-regulated gene expression in NMuMG cells, we have shown regulation of multiple genes that play important roles in cell cycle control and EMT. In addition, we have identified several novel TGF-beta-regulated genes that may mediate previously unknown TGF-beta functions.

Acetyltransferase p300 (KAT3B) plays key roles in signaling cascades that support cancer cell survival and sustained proliferation. p300 is overexpressed in several types of cancer, which correlates with tumor progression. Thus, p300 represents a rational anticancer therapeutic target. To discover novel anticancer agents that target p300, we conducted a high-throughput screening campaign. A library of 622,079 compounds was assayed for cytotoxicity to the triple-negative breast cancer (TNBC) cell line MDA-MB-231 but not to the human mammary epithelial cells. The resulting compounds were tested in a biochemical assay for inhibiting the enzymatic activity of p300. One compound (L002, NSC764414) displayed an IC50 of 1.98 μM against p300 in vitro, inhibited acetylation of histones and p53, and suppressed STAT3 activation in cell-based assays. L002 could be docked to the active site of the p300 catalytic domain. Biochemical tests of a series of related compounds revealed functional groups that may impact inhibitory potency of L002 against p300. Interestingly, these analogs showed inhibitory activities against CBP (the cellular paralog of p300), PCAF and GCN5, but not to other acetyltransferases (KAT5, KAT6B and KAT7), histone deacetylases (HDACs) and histone methyltransferases. Among the NCI-60 panel of cancer cell lines, leukemia and lymphoma cell lines were extremely sensitive to L002, whereas it is toxic to only a limited number of cell lines derived from solid tumors. Notably, breast cancer cell lines, especially those derived from TNBC, were highly susceptible to L002. In vivo, it potently suppressed tumor growth and histone acetylation of MDA-MB-468 xenografts. Thus, these new acetyltransferase inhibitors are potential anticancer therapeutics with specific activities against breast cancer and hematological malignancies. This abstract is also presented as Poster B43. Citation Format: Heng Yang, Christie E. Pinello, Jian Luo, Dawei Li, Yunfei Wang, Zhao Y. Lisa, Stephan C. Jahn, S. Adrian Saldanha, Jamie Planck, Kyla R. Geary, Haiching Ma, Brian K. Law, William R. Roush, Peter Hodder, Daiqing Liao. A new class of small molecule acetyltransferase inhibitors discovered through high-throughput screening are potent anticancer agents with cancer-type specific activity. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr PR16.

The p53-dependent tetraploidy checkpoint serves to block the proliferation of cells that double their genome without dividing. Oftentimes, cancers will contain a tetraploid sub-population, indicating either an ability to bypass this checkpoint or that the checkpoint is no longer functional. In studying the chromosomal instability of tumor cell lines from mice with mammary expression of a constitutively active Cyclin D1-Cdk2 fusion protein (D1K2), we noticed an increased rate of polyploidization in the presence of the spindle-poison paclitaxel. This was recapitulated in an MCF10A cell line model, and it was discovered that this is due to the inability of the tetraploidy checkpoint to completely stop DNA replication in tetraploid cells with constitutively active Cdk2. Biochemical analysis shows that tetraploid cells expressing D1K2 maintain Rb phosphorylation longer than tetraploid control cells, matching the phenotype seen in cells that have p53 stably knocked down. Further DNA replication, yielding octaploid cells, appears to further intensify the tetraploidy checkpoint, arresting the cells. Due to the fact that approximately 50% of all breast cancers overexpress Cyclin D, these observations raise intriguing questions about the treatment of tumors with overactive Cdk2. While it appears these tetraploid MCF10A cells are unviable, we can not account for other mutations that may exist in vivo and allow the proliferation of a chromosomally unstable tetraploid tumor population. Citation Format: Stephan Jahn, Patrick Corsino, Mary Law, Brad Davis, Brian Law. Overactive Cdk2 permits polyploidy by overwhelming the tetraploidy checkpoint. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 571. doi:10.1158/1538-7445.AM2013-571

The classic chemotherapeutic, paclitaxel, causes endoreduplication in cells, but the mechanism and cellular characteristics that promote this genome doubling are poorly understood. In studying the chromosomal instability of tumor cell lines from mice with mammary expression of a constitutively active Cyclin D1-Cdk2 fusion protein, we noticed an increased rate of this paclitaxel induced endoreduplication. Further studies have shown that this effect is specifically due to the kinase activity of Cdk2 and that increased Cdk2 activity causes of upregulation of the mitotic checkpoint protein, Mad2. While increased Mad2 expression alone has previously been shown to cause mitotic defects and genome doubling, it does not appear to be the culprit in this situation. We instead found that normalization of Mad2 levels through short hairpin RNA-mediated knockdown further increased the rate of endoreduplication in the presence of paclitaxel. It was seen that while exposure to the spindle poison further elevated Mad2 levels, long-term treatment led to an overall decrease in protein expression. This presumably allows cells to escape mitotic arrest and enter G1 phase, potentially triggering the somewhat controversial p53-mediated tetraploidy checkpoint. Indeed, stable knock-down of p53 cooperates with increased Cdk2 activity to create a very high rate of endoreduplication. These findings raise intriguing questions about the consequences of using one of the world's most widely used chemotherapeutic agents in patients with increased Cdk2 activity. Future studies will investigate whether this increased rate of endoreduplication worsens treatment outcome by eliciting chromosomal instability or improves it by generating cells that are unviable in the long-term. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A71.

mTORC1 inhibitors such as rapamycin and its analogs (rapalogs) are promising anticancer agents, which are effective against some tumors, yet ineffective against others. Understanding the mechanisms controlling tumor responsiveness or resistance to rapalogs is therefore critical. Much of the anticancer activity of mTORC1 inhibitors stems from inhibition of cancer cell proliferation and suppression of the endothelial cell proliferation required for angiogenesis. Rapalogs induce a G1 cell cycle arrest, which has been attributed to suppression of the translation of proliferation-associated proteins. However the exact mechanism by which mTORC1 inhibition results in G1 cell cycle arrest is unclear. Since activation of E2F-dependent transcription is required for the G1/S-phase cell cycle transition, we examined the mechanisms by which rapamycin inhibits the expression of E2F1, which is itself encoded by an E2F-dependent gene. Rapamycin treatment decreased the binding of Cdk2 to the E2F sites of the E2F1 promoter in DNA oligonucleotide affinity purification experiments. This observation was corroborated by a decrease in Cdk2 binding to E2F4, p130, and p107, and a corresponding increase in Cdk2 binding to p27 in coimmunoprecipitation experiments. This suggests a model in which Cdk2 is recruited to E2F sites of promoters by complex formation with E2F4, p130, and p107 and functions to relieve the repression of these elements by maintaining p130 and p107 in their hyperphosphorylated forms. Consistent with this model, rapamycin treatment decreased kinase activity associated with E2F-DNA binding elements, but had little effect on overall Cdk2 activity in crude cell lysates. Rapamycin caused disruption of Cdk2/p130-, p107-, E2F4-containing complexes on E2F promoter elements. These results provide insights into how rapamycin treatment inhibits E2F-dependent transcription. It is hoped that further analysis of Cdk2/p130/E2F complexes will reveal mechanisms of tumor resistance to rapalogs and opportunities for optimizing the efficacy of mTORC1 inhibitors using combination therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2951. doi:10.1158/1538-7445.AM2011-2951

HLM006474 is a recently identified small molecule E2F inhibitor. Since E2F is known to drive expression of genes required for cell cycle progression, it is predicted that blocking the E2F pathway will slow proliferation and have anticancer activity. Microarray analysis demonstrates that HLM006474 treatment results in profound changes in the expression of genes involved in cell cycle progression and nucleotide biosynthesis. Flow cytometry reveals that treatment of A375 cells results in a cell cycle arrest, primarily in G2/M. As would be expected, cell lines deficient in Rb are more sensitive to E2F inhibition than are syngeneic lines expressing Rb. HLM006474 reduces the proliferation of a broad array of cell lines with a biological IC50 that varies between 5 and 70 μM. Combination of HLM006474 with common chemotherapeutic agents cisplatin and gemcitabine demonstrate little synergy; however HLM006474 synergizes with paclitaxel and a novel cdk inhibitor NSC11702. Interestingly, we discovered that paclitaxel sensitivity inversely correlated with E2F4 levels in a series of non-small cell lung cancer (NSCLC) lines, suggesting that synergy between HLM006474 and paclitaxel is due to HLM006474's inhibition of E2F4 activity and expression. To test this, H1299 cells which have high E2F4 levels and a high paclitaxel IC50 were depleted of E2F4 with siRNA and the IC50 determined. Taken together, these results suggest that HLM006474 may have efficacy against cancers in which E2F is deregulated and may be useful in combination with other drugs that target cell cycle components. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2943. doi:10.1158/1538-7445.AM2011-2943

Angiogenesis is an essential component of tumour development and is tightly regulated by a network of pro-angiogenic and anti-angiogenic factors. The tumour cells are known to produce many of the proteins that interact with vascular endothelial cells and drive the angiogenic process. Of the known angiogenic factors, VEGF has been established as a potent inducer of tumor angiogenesis. Like almost all secretory and membrane proteins, VEGF attains its final folded or assembly conformation in ER before making its way to its target organelles or the cell surface. The deleterious tumor microenvironment characterized by progressive hypoxia, nutrient starvation and acidosis results in the ER stress in the tumor vascular endothelial cells, which in turn leads to the unfolded/misfolded VEGF accumulated in the ER. Molecular chaperon proteins counteract the formation of aberrantly folded VEGF and allow its correct folding. One of molecular chaperons is CRYAB. It has been reported that CRYAB contains interactive sequences for VEGF. The misfolded VEGF interacts with BiP (chaperone immunoglobulin heavy chain-binding protein) causing its release from one or more of the three ER stress sensors [PKR-like ER kinase (PERK), inositol-requiring protein-1 (IRE1) and transcription factor 6 (ATF6)], which leads to activation of the unfolded protein response (UPR) pathway. All three sensor pathways can lead to chaperone induction but the IRE1 and ATF6 pathways are thought to play the major role in up-regulation of CRYAB. Activation of IRE1 generates a mature XBP-1 mRNA by unconventional splicing of XBP-1 pre-mRNA and activated XBP-1 up-regulates CRYAB. Activation of ATF6 results in its translocation to the Golgi apparatus and proteolytic cleavage to yield its cytosolic domain not only for directly up-regulating genes encoding CRYAB and folding catalysts, but also for assisting IRE1-dependent upregulation of CRYAB. An excessively elevated level of CRYAB has been detected in breast carcinomas, correlating with lymph node involvement resulting in metastasis and short term survival. Taken together, it is clear that CRYAB has the potential to a) protect VEGF from intracellular degradation thus maintaining the intracellular pool and b) increase VEGF secretion. Both these actions serve to enhance the autocrine VEGF signaling response which serves as one of strategies adapted by the tumor vasculature to perpetuate aberrant angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-4. doi:10.1158/1538-7445.AM2011-LB-4

The Epithelial to Mesenchymal Transition (EMT), by facilitating the metastatic process, is an important step in tumor progression for many types of cancer. We have developed a mouse tumor model, driven by the HER2/neu oncogene, whereby this process can be studied robustly both in vivo and in cell culture. This system has revealed significant changes in mitogenic signaling networks that occur during EMT. Post-EMT cancer cells acquired increased responsiveness to HGF, PDGF, and LPA. This change in responsiveness was accompanied by a decrease of Her2, c-Src and IRS-1 levels, as well as an increase in Axl, PDGFR and c-Met levels. Perturbations in these signaling cascades results in the differential sensitivity to certain anti-cancer therapeutics. Post-EMT cells were less sensitive than Pre-EMT cells to the MEK inhibitor U0126. Furthermore, while Pre-EMT cells exhibited cell cycle arrest in response to rapamycin, Post-EMT cell proliferation was stimulated. These observations may have significant bearing on the efficacy of targeted therapeutic agents in patients with metastatic breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2895. doi:10.1158/1538-7445.AM2011-2895