Yao Dai

Development of optimized AAV3 serotype vectors: Mechanism of high-efficiency transduction of human liver cancer cells

Our recent studies have revealed that among the 10 different commonly used adeno-associated virus (AAV) serotypes, AAV3 vectors transduce human liver cancer cells extremely efficiently because these cells express high levels of human hepatocyte growth factor receptor (hHGFR), and AAV3 utilizes hHGFR as a cellular co-receptor for viral entry. In this report, we provide further evidence that both extracellular as well as intracellular kinase domains of hHGFR are involved in AAV3 vector entry and AAV3-mediated transgene expression. We also document that AAV3 vectors are targeted for degradation by the host cell proteasome machinery, and that site-directed mutagenesis of surface-exposed tyrosine (Y) to phenylalanine (F) residues on AAV3 capsids significantly improves the transduction efficiency of Y701F, Y705F and Y731F mutant AAV3 vectors. The transduction efficiency of the Y705+731F double-mutant vector is significantly higher than each of the single mutants in liver cancer cells in vitro. In immunodeficient mouse xenograft models, direct intratumoral injection of AAV3 vectors also led to high-efficiency transduction of human liver tumor cells in vivo. We also document here that the optimized tyrosine-mutant AAV3 vectors lead to increased transduction efficiency following both intratumoral and tail-vein injections in vivo. The optimized tyrosine-mutant AAV3 serotype vectors containing proapoptotic genes should prove useful for the potential gene therapy of human liver cancers.

BMS-777607, a Small-Molecule Met Kinase Inhibitor, Suppresses Hepatocyte Growth Factor–Stimulated Prostate Cancer Metastatic Phenotype In vitro

Most prostate cancer–related deaths are due to advanced disease with patients with metastatic prostate cancer having a 5-year survival rate of only 34%. Overexpression of c-Met receptor tyrosine kinase has been highly associated with prostate cancer progression and metastasis. In the present studies, the effect of BMS-777607, a selective and potent small-molecule Met kinase inhibitor that has been advanced to clinical evaluation, on hepatocyte growth factor (HGF)–mediated cell functions and signaling pathways was evaluated in c-Met–expressing PC-3 and DU145 prostate cancer cells. BMS-777607 treatment had little effect on tumor cell growth but inhibited cell scattering activated by exogenous HGF, with almost complete inhibition at 0.5 μmol/L in PC-3 and DU145 cells. This agent also suppressed HGF-stimulated cell migration and invasion in a dose-dependent fashion (IC50 < 0.1 μmol/L) in both cell lines. Mechanistically, nanomolar doses of BMS-777607 potently blocked HGF-stimulated c-Met autophosphorylation and downstream activation of Akt and extracellular signal-regulated kinase. In addition, both wortmannin and U0126, but not dasatinib, attenuated cell scattering and migration induced by HGF, suggesting the involvement of the phosphoinositide 3-kinase and mitogen-activated protein kinase pathways, but not of Src or focal adhesion kinase, in HGF-mediated motogenic effects. Taken together, these data indicate that the downregulation of c-Met signaling by BMS-777607 treatment can significantly disrupt key steps in the metastatic cascade, suggesting that such a targeting strategy may hold promise for the treatment of advanced prostate cancer. Mol Cancer Ther; 9(6); 1554–61. ©2010 AACR.

Constitutively active c-Met kinase in PC-3 cells is autocrine-independent and can be blocked by the Met kinase inhibitor BMS-777607.

BackgroundThe c-Met receptor tyrosine kinase is aberrantly activated in many solid tumors. In a prior study we showed that prostate cancer PC-3 cells exhibit constitutively activated c-Met without exogenous hepatocyte growth factor (HGF); however whether this characteristic is due to an endogenous HGF/c-Met autocrine loop remains controversial. In the current study we examined the response of PC-3 cells to an anti-HGF neutralizing antibody or a small molecule Met kinase inhibitor (BMS-777607).MethodsCell scattering was tested by monitoring cell morphology after HGF stimulation. Cell migration was examined by both “wound-healing” and transwell assasy and invasion was detected by Matrigel-coated transwell assay. Proliferation, survival and anoikis were determined by MTT, colony formation and trypan blue exclusion assay, respectively. Gene and protein expression were assessed by real-time PCR and Western blot, respectively.ResultsAlthough HGF mRNA could be detected in PC-3 cells, the molecular weight of secreted “HGF” protein was inconsistent with the functional recombinant HGF. Furthermore, conditioned medium from PC-3 cell cultures was ineffective at triggering either motogenic behavior or c-Met signaling in DU145, another prostate cancer cell line expressing c-Met but lacking basal c-Met activation. PC-3 cells also were not responsive to the anti-HGF neutralizing antibody in experiments assessing proliferation, migration, or c-Met signaling. BMS-777607 treatment with micromolar doses nonetheless led to significant inhibition of multiple PC-3 cell functions including proliferation, clonogenicity, migration and invasion. At the molecular level, BMS-777607 suppressed autophosphorylated c-Met and downstream c-Src and Akt pathways.ConclusionsThese results suggest that the constitutive c-Met activation in PC-3 is independent of autocrine stimulation. Because PC-3 cells were responsive to BMS-777607 but not the anti-HGF antibody, the findings also indicate that under circumstances where c-Met is constitutively hyperactive in the absence of functional HGF, targeting the c-Met receptor remains a viable therapeutic option to impede cancer progression.

Hypoxia, Metastasis, and Antiangiogenic Therapies

It is well known that aberrant microenvironmental conditions in solid tumors negatively impact their response to conventional anticancer therapies. Indeed the presence of hypoxia in the primary tumor has been identified as a prognostic indicator of treatment outcome in a number of clinical settings. Hypoxic conditions can arise at the limits of oxygen diffusion from blood vessels (diffusion or chronic hypoxia) or as a consequence of vascular collapse (perfusion or acute hypoxia). Both types of hypoxia upregulate key signaling pathways associated with metastasis and related tumor cell behavior and function; and although the impact of acute and chronic oxygen deprivation on these pathways may differ markedly, the effect of such exposures is to significantly enhance the metastatic phenotype. In lieu of the critical role of metastasis in treatment failures, there has been significant interest in developing molecular targeting strategies that impair signaling pathways associated with tumor cell dissemination. Many of these targeted tumor cell associated functions are enhanced by hypoxia, including invasion and angiogenesis. Agents targeting angiogenesis are of interest because they could interfere with the progression of established microtumor deposits to macroscopic size by interfering with blood vessel development but they also raise the caveat that the use of such agents might induce hypoxia in the primary tumor which could conceivably enhance the dissemination of tumor cells. This chapter discusses the impact of oxygen deficiencies on the maintenance of stem-like characteristics of tumor initiating cells, the potential interplay between genomic markers and hypoxia/HIFs, and the effect of hypoxia on signaling pathway critical to the metastatic process, including VEGF, c-Src, and the HGF/c-Met axis. It further explores how the interference with signaling molecules may have significant therapeutic potential in the development of novel therapeutic intervention approaches for the treatment of cancer metastasis.

Abstract B013: Combined molecular targeting Src and mTOR pathways to impair prostate cancer cell survival

Overexpression of oncoproteins Src and mTOR has been shown to be highly associated with prostate cancer progression and poor clinical outcomes; therefore, small-molecule Src or mTOR inhibitors are being extensively evaluated in clinical trials for advanced prostate cancer. However, clinical data suggest that single treatment with any of these inhibitors only yields limited effects in patients. One possibility is that redundant signaling pathways in tumor cells exist that allow tumor cell uninterrupted upon blockade of purely one pathway. We hypothesize that Src and mTOR are two compensatory pathways that may cooperate to promote prostate cancer survival. Here we studied treatment effects of combining inhibitors targeting Src (saracatinib) and mTOR (rapamycin) on functional behaviors associated with tumor growth in prostate cancer cells with high level expression of both kinases. Combination of both inhibitors was found to inhibit cell proliferation and clonogenic survival more strongly than either single agent, as assessed by MTT and colony formation assay, respectively. Western blot analysis demonstrated that co-targeting Src and mTOR kinases leads to not only inhibition on phosphorylation of these oncoproteins but also greater blockade of multiple downstream pathways such as focal adhesion kinase (FAK), Akt, ERK, S6K, and 4E-BP1. These data indicate that combined application of small-molecule inhibitors targeting two different key pathways may cause significant impairment of phenotypes associated with prostate cancer growth and survival, suggesting that this combinative intervention modality may serve as a potential anticancer strategy that could be further tested in advanced prostate cancer models in vivo. Citation Format: Yao Dai, Dietmar W. Siemann. Combined molecular targeting Src and mTOR pathways to impair prostate cancer cell survival [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B013.

Abstract 2792: Impact of hypoxia on tyrosine kinase activation in cancer cells

Purpose: Metastasis is the major cause of therapeutic failure and high mortality in cancer patients. Growing evidence has demonstrated that dysregulated intracellular tyrosine kinase (TK) activity can play an important role in promoting disease onset and progression, while low oxygen tensions (hypoxia) are a typical external driving force that facilitates tumor cell dissemination. However, whether hypoxia-induced metastasis is mediated by TK-signaling is largely unknown. In this study, we have identified potential TK(s) that can mediate hypoxia-induced cell functions associated with metastasis, and further determine the therapeutic potential of small molecule TK inhibitors in intervening these functional behaviors. Methods: Cancer cells were exposed to low oxygen tensions (1% O2) for varied durations (0, 2, 6, 24 h), with or without treatment of desired molecular agents. To test cellular phenotypes, cells were seeded into appropriate culture vehicles and cell migration and invasion will be examined by scratch and transwell-based assays. At the molecular level, pan-TKs phosphorylation in malignant versus normal cells, as well as in cancer cells with or without hypoxic exposure were detected by Western blotting. To further investigate the association of p-Src and HIF-1α in patient tissues, tissue microarray (TMA) with prostate cancer patients was conducted by immunohistochemical staining followed by independent quantification on the H-score of the two proteins. Results: TK phosphorylation in cancer cells is generally stronger than in normal cells. Similar observation is found in patient tumor tissues versus adjacent normal counterparts. When exposing cells to hypoxia, short term hypoxic exposure (2-6 h) generally induces greater cell migration and invasion than prolonged hypoxic exposure (24 h). Such functional behaviors tend to be associated with elevated tyrosine kinome activation, although the exact TKs in different cell lines may vary. Subsequent kinome screening reveals that the well-known oncoprotein c-Src is activated under particularly acute hypoxia. Further, small molecule inhibitors targeting these TKs have shown stronger inhibition on cell migration and invasion under hypoxia than normoxia. Finally, TMA analysis suggests that p-Src expression is significantly associated with HIF-1α particularly in prostate cancer patients with advanced disease. Conclusion: The data reveal that short term hypoxia is able to enhance metastasis-associated phenotypes by activating a group of TKs such as p-Src. In addition, Src inhibitors are showing higher potential in impeding hypoxia-induced functional behaviors. These findings suggest that at least in some cancers over-activated TKs can be used as indirect markers for hypoxia and more importantly, molecular agents targeting hypoxia-activated TKs may hold therapeutic potential to prevent metastatic spread of cancer cells that have experienced particularly short-term oxygen depletion. Citation Format: Yao Dai, Dietmar Siemann. Impact of hypoxia on tyrosine kinase activation in cancer cells. [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 2792.

Abstract B59: Dual mTOR kinase inhibitor reverses rapamycin resistance in prostate cancer cells

The mTOR pathway is frequently over-activated in human cancers. However, classic allosteric mTOR inhibitors rapamycin and its analogs only exert limited clinical benefits in patients. It has been shown that many tumors either fail to respond to rapamycin initially or are able to acquire resistance after the initial treatment of rapamycin. Such primary and secondary resistances remain as major concerns in rapamycin-based anti-cancer therapies that may inevitably lead to therapeutic failure. We have previously shown that the ATP-competitive dual mTOR kinase inhibitor AZD8055 can significantly inhibit malignant behaviors in parental cells however its functional efficacies in the rapamycin-resistance setting remains unclear. Cancer cells with wildtype PTEN were selected as models of primary rapamycin resistance since the mTOR pathway is usually less abnormal in these cells. To establish cells with acquired rapamycin resistance, PTEN-null prostate cancer PC-3 cells were gradually treated with increasing concentrations of rapamycin (up to 10 μM) until the cells were completely unresponsive to rapamycin (PC-3RR). Cell proliferation was determined by MTT assay and clonogenic survival was tested by colony formation assay. m7G pull-down assay was employed to detect the cap-dependent translation. mTOR downstream pathways were detected by Western blotting. PTEN wildtype and PC-3RR cells essentially failed to respond to rapamycin; IC50 values were greater than 100 nM in PTEN wildtype cells and 1 μM in PC-3RR cells, respectively. However, for both cell types AZD8055 suppressed proliferation at the nanomolar level in a dose dependent manner, with IC50 values less than 30 nM. Similarly, cellular clonogenic capacity was impaired by AZD8055 to a greater extent than was the case for rapamycin. At the molecular level, AZD8055, but not rapamycin, strongly prevents the dissociation of 4E-BP1 from eIF4E, therefore blocking cap-dependent translation. Further, while the p-S6K (T389) and p-S6 (S235/236) pathway was potently inhibited by both agents, AZD8055 displayed more striking inhibition on p-4E-BP1 (T37/46) and p-Akt (S473) than did rapamycin in all cells tested. Interestingly, similar to rapamycin, AZD8055 slightly increased p-Akt (T308), suggesting the existence of the PI3K-mediated feedback loop. However, the PI3K inhibitor wortmannin failed to sensitize cell response to AZD8055, implying such feedback-dependent activation has little impact on the drug efficacy. These findings indicate that dual mTOR kinase inhibitor AZD8055 significantly inhibits malignant functions in cancer cells that are highly resistant to rapamycin. Such effects are likely to be associated with deactivation of rapamycin-insensitive mTOR downstream components such as Akt and 4E-BP1, but not the upstream feedback pathway. These data provide in vitro evidence in support of future evaluation of ATP-competitive mTOR inhibitors in cancer models that are resistant to rapamycin analogs. Citation Format: Yao Dai, Li Zhao, Dietmar W. Siemann. Dual mTOR kinase inhibitor reverses rapamycin resistance in prostate cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B59.

Abstract 5507: Activation of a subset of growth factor-associated genes in cetuximab-sensitive colorectal cancer GEO cells with KRAS mutation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA KRAS mutation has been widely used in the clinic as a predictive biomarker in metastatic colorectal cancer (mCRC) for insensitivity to the anti-EGFR antibody cetuximab (Erbitux). However recent clinical studies indicate that a subset of mCRC patients with mutated KRAS may still retain sensitive to the drug. Thus it is important to identify alternative signaling pathways that bypass the activated KRAS pathway and mediate cetuximab-induced tumor killing effects. The mCRC cell line GEO harbors mutated KRAS (G12A) but is one of the most sensitive cell lines to cetuximab. The underlying mechanisms by which KRAS-mutated cells remain responsive to EGFR inhibitors have not been explored. In the current study, we compared the expression of KRAS and EGFR in four CRC cells (GEO, LOVO, HT-29, Caco2) with varying KRAS status and evaluated the treatment effects of cetuximab in these models. Cell proliferation was detected by MTT assay. Drug effects in xenografts were assessed by monitoring tumor growth delay. At the molecular level, active K-Ras was detected by pull-down assay and EGFR-associated signaling pathways were determined by Western blotting. KRAS gene expression was tested by qPCR, and EGFR expression in tumor tissues was tested by immunohistochemical staining. Finally, GEO xenografted tumors with or without cetuximab treatment were processed for PCR array analysis to detect the gene expression of molecular components associated with growth factor (EGF, PDGF) signaling networks. In accordance with the sequencing information, we confirmed the constitutively active K-Ras in GEO cells compared to KRAS-wildtype counterparts. However, consistent with other studies, we found that of GEO cell proliferation and xenograft tumor growth were significantly inhibited by cetuximab, a phenotype similar to that in KRAS normal HT-29 but not observed in the KRAS mutated LOVO model. Further, cetuximab inhibited expression of p-Akt but not p-ERK, p-STAT3, p-Src and total EGFR. Interestingly, PCR arrays showed that a subset of genes was highly activated in cetuximab treated GEO tumors, including LTA, MAP2K7, MAPK10 and STAT1. These gene products have been previously implicated in growth arrest and apoptosis however their association with EGFR/K-Ras signaling remains unknown. Studies on examining the functional role of these candidate genes in mCRC cells in response to cetuximab are ongoing. These preliminary findings support further investigations to identify alternative molecular signatures as potential biomarkers that may predict cetuximab-mediated tumor response in a subset of mCRC cells that harbors KRAS mutation. Citation Format: Yao Dai, Dietmar Siemann, Wenyin Shi. Activation of a subset of growth factor-associated genes in cetuximab-sensitive colorectal cancer GEO cells with KRAS mutation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5507. doi:10.1158/1538-7445.AM2014-5507

Abstract A5: Functional role of SOX2 in hypoxia-mediated metastasis-associated functions in prostate cancer

Purpose: SOX2 is a typical embryonic stem cell marker that is involved in not only tumorigenesis but also metastasis in several human malignancies including prostate cancer. Hypoxia has been demonstrated as a common feature of prostate cancer that is associated with disease progression and poor prognosis. Although SOX2 has been shown to be activated by oxygen depletion in other cancer types, its functional role under hypoxic microenvironment in prostate cancer setting and its association with hypoxia inducible factors (HIFs) have not been reported. In the present study, we evaluate the role of SOX2 in hypoxia-mediated cellular behaviors associated with metastasis, including invasion and sphere formation, in prostate cancer cells. Experimental Design: A tissue microarray containing samples from 80 prostate cancer patients was employed to examine the expression of HIF-1α and its correlation with SOX2 by immunohistochemical analysis. Expression of stem cell markers and HIFs was detected by Western blot. The role of SOX2 and HIF-1/2α in the regulation of cell invasion and sphere formation with various hypoxic exposures was investigated by short hairpin RNA (shRNA) - mediated knockdown in PC-3, DU145 and LNCaP cells. Results: HIF-1α expression was significantly elevated in prostate cancer compared to non-malignant prostate tissues, and in tumors HIF-1α was strongly correlated with SOX2. In all three prostate cancer cells, while HIF-1α and HIF-2α were mainly induced under short term (acute) and long term (chronic) hypoxic conditions, respectively, SOX2 was the only one among five stem cell markers that was stimulated by both hypoxic exposures. Genetic depletion of SOX2 attenuated capacities of tumor cells to invade under acute hypoxia and form spheroids under chronic hypoxia. In addition, knockdown of HIF-1α but not HIF-2α decreased acute hypoxia-mediated cell invasion, whereas only HIF-2α gene silencing resulted in reduced sphere formation capacity mediated by long term oxygen deprivation. Conclusions: SOX2 is suggested to be correlated with hypoxia in prostate cancer. SOX2 plays an important role in acute hypoxia-induced invasiveness and chronic hypoxia-induced stemness that is associated with HIF-1α and HIF-2α activation, respectively. These results indicate that SOX2 may be a key mediator for hypoxia-induced metastasis-associated functions hence may serve as a potential target for therapeutic interventions for advanced prostate cancer. Citation Format: Yao Dai, Kyung-Mi Bae, Johannes Vieweg, Dietmar W. Siemann. Functional role of SOX2 in hypoxia-mediated metastasis-associated functions in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A5.

Abstract 3761: Efficacy of the ATP-competitive mTOR inhibitor AZD8055 in PTEN-wild type cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Although the PI3K/Akt/mTOR pathway is frequently hyperactivated in human cancers due to the loss of function of the PTEN tumor suppressor, a proportion of cancer cells still carries wild type PTEN and thus the PI3K/Akt/mTOR pathway in these cells may remain unaltered. While it has been widely accepted that these cancer cells are less susceptible to the allosteric mTOR inhibitor rapamycin, it is unclear whether this is also the case for an ATP-competitive mTOR inhibitor. To address this question we evaluated the effects of the mTOR kinase inhibitor AZD8055 in a panel of wild type PTEN expressing cancer cells (6 cell lines of 4 tumor types). The phosphorylated Akt (S473) level was undetectable in most of these cells, suggesting the regular mTOR complex 1 (mTORC1) pathway and low mTOR complex 2 (mTORC2) activity. Cell proliferation was determined by WST-1 (MTT) assay and clonogenic survival was tested by colony formation assay. AZD8055 suppressed cell proliferation at the nanomolar level in a dose dependent manner, with IC50 values in a range of 6∼35 nM for different cell types. In contrast, rapamycin exhibited limited anti-proliferative effect, with less than 50% of inhibition observed even with higher doses in most cell lines. Similarly, clonogenic capacity was impaired by AZD8055 to a greater extent than was the case for rapamycin. To detect the mTOR pathway alterations by drug treatment, two cell lines with different expression levels of mTORC1 downstream pathways were selected: murine mammary tumor 4T1 cells (low phosphorylated S6 and high phosphorylated 4E-BP1) and human breast cancer MDA-MB-231 cells (normal phosphorylated S6 and moderate phosphorylated 4E-BP1). While the phospho-S6K (T389) and phospho-S6 (S235/236) pathway was potently inhibited by both agents, AZD8055 displayed more striking inhibition of the hierarchical 4E-BP1 phosphorylation (T37/46, T70 and S65) than rapamycin in both cells. In addition, both rapamycin and AZD8055 slightly increased Akt phosphorylation (T308 or S473), suggesting that the activation of the feedback loop controlled by S6K was functional. However, the PI3K inhibitor wortmannin did not sensitize cells to either agent, implying such feedback-dependent activation is dispensable for attenuating the drug response. Taken together, these findings indicate that AZD8055 significantly inhibits growth and survival of cancer cells that have wild type PTEN and normal PI3K/Akt/mTOR signaling, an observation mainly associated with the comprehensive activation of 4E-BP1. These data provide in vitro evidence in support of broadening the application of ATP-competitive mTOR inhibitors in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3761. doi:1538-7445.AM2012-3761