Sazzad Hassan

Behavioral stress accelerates prostate cancer development in mice

Prostate cancer patients have increased levels of stress and anxiety. Conversely, men who take beta blockers, which interfere with signaling from the stress hormones adrenaline and noradrenaline, have a lower incidence of prostate cancer; however, the mechanisms underlying stress-prostate cancer interactions are unknown. Here, we report that stress promotes prostate carcinogenesis in mice in an adrenaline-dependent manner. Behavioral stress inhibited apoptosis and delayed prostate tumor involution both in phosphatase and tensin homolog-deficient (PTEN-deficient) prostate cancer xenografts treated with PI3K inhibitor and in prostate tumors of mice with prostate-restricted expression of c-MYC (Hi-Myc mice) subjected to androgen ablation therapy with bicalutamide. Additionally, stress accelerated prostate cancer development in Hi-Myc mice. The effects of stress were prevented by treatment with the selective β2-adrenergic receptor (ADRB2) antagonist ICI118,551 or by inducible expression of PKA inhibitor (PKI) or of BCL2-associated death promoter (BAD) with a mutated PKA phosphorylation site (BADS112A) in xenograft tumors. Effects of stress were also blocked in Hi-Myc mice expressing phosphorylation-deficient BAD (BAD3SA). These results demonstrate interactions between prostate tumors and the psychosocial environment mediated by activation of an adrenaline/ADRB2/PKA/BAD antiapoptotic signaling pathway. Our findings could be used to identify prostate cancer patients who could benefit from stress reduction or from pharmacological inhibition of stress-induced signaling.

Protein Kinase D1 Suppresses Epithelial-to-Mesenchymal Transition through Phosphorylation of Snail

Cancer cells undergo epithelial-mesenchymal transition (EMT) as a program of increased invasion and metastasis during cancer progression. Here, we report that a novel regulator of EMT in cancer cells is protein kinase D1 (PKD1), which is downregulated in advanced prostate, breast, and gastric cancers. Ectopic reexpression of PKD1 in metastatic prostate cancer cells reversibly suppressed expression of mesenchyme-specific genes and increased epithelial markers such as E-cadherin, whereas small interfering RNA-mediated knockdown of PKD1 increased expression of mesenchyme markers. Further, PKD1 inhibited tumor growth and metastasis in a tumor xenograft model. PKD1 phosphorylates Ser(11) (S11) on transcription factor Snail, a master EMT regulator and repressor of E-cadherin expression, triggering nuclear export of Snail via 14-3-3σ binding. Snail S11 mutation causes acquisition of mesenchymal traits and expression of stem cell markers. Together, our results suggest that PKD1 functions as a tumor and metastasis suppressor, at least partly by regulating Snail-mediated EMT, and that loss of PKD1 may contribute to acquisition of an aggressive malignant phenotype.

BAD Dephosphorylation and Decreased Expression of MCL-1 Induce Rapid Apoptosis in Prostate Cancer Cells

PTEN loss and constitutive activation of the PI3K signaling pathway have been associated with advanced androgen-independent prostate cancer. PTEN-deficient prostate cancer C42Luc cells survive in serum-free media and show relative resistance to apoptosis even in the presence of the PI3K inhibitor ZSTK474. Yet, when ZSTK474 is combined with the translation inhibitor cycloheximide, C42Luc cells undergo apoptosis within 6 hours. We identified dephosphorylation of BAD (Bcl2-associated death promoter) as a main apoptosis-regulatory molecule downstream from PI3K, and loss of MCL-1 (Myeloid cell leukemia -1) as a major target of cycloheximide. The combination of MCL-1 knockdown and expression of phosphorylation-deficient mutant BAD2SA is sufficient to trigger rapid apoptosis in prostate cancer cells. These results establish the mechanism for the synergistic induction of apoptosis by the combination of a PI3K inhibitor and of a protein synthesis inhibitor in PTEN-deficient prostate cancer cells.

Surgical Stress Delays Prostate Involution in Mice

Androgens control growth of prostate epithelial cells and androgen deprivation induces apoptosis, leading to prostate involution. We investigated the effects of surgical stress on prostate involution induced by androgen ablation and determined the underlying mechanisms. Androgen ablation in mice was induced by surgical castration and administration of the anti-androgenic drugs bicalutamide and MDV3100. Surgical stress was induced by sham castration under isoflurane anesthesia. Surgical stress delayed apoptosis and prostate involution induced by anti-androgenic drugs. These effects of stress were prevented by administering the selective beta2-adrenoreceptor antagonist ICI118,551 and were also blocked in BAD3SA/WT mice expressing phosphorylation-deficient mutant BAD3SA. These results indicate that apoptosis and prostate involution in response to androgen ablation therapy could be delayed by surgical stress via the beta2-adrenoreceptor/BAD signaling pathway. Thus, surgery could interfere with androgen ablation therapy, whereas administration of beta2-adrenoreceptor antagonists may enhance its efficacy.

Animal Model: Xenograft Mouse Models in Esophageal Adenocarcinoma

Researchers often use murine models of esophageal cancer to evaluate novel therapies prior to clinical protocol treatment. Subcutaneous xenograft models are often used for testing the efficacy of anticancer agents in many cancers including esophageal adenocarcinoma. However, mice subcutaneous esophageal adenocarcinoma models only represent local tumor growth and do not provide any information about a survival benefit for a particular anticancer regimen, which is very crucial for experimental treatment efficacy. In addition, anticancer agents may well inhibit subcutaneous tumor growth without effecting overall animal survival. Herein, we describe a peritoneal dissemination mouse xenograft model for survival outcome analysis with intraperitoneal injection of human esophageal adenocarcinoma cell lines.

Abstract 4826: Synergistic effects of foretinib with lapatinib in MET and HER2 co-activated experimental esophageal adenocarcinoma

Introduction: Recent studies have demonstrated that HER2 and MET receptor tyrosine kinases are co-overexpressed in a subset esophageal adenocarcinoma (EAC). We therefore studied the usefulness of combining HER2 and MET targeting by small-molecule inhibitors foretinib and lapatinib both in-vitro and in-vivo models of experimental EAC. Methods: In this study we first characterized MET and HER2 activation in a panel of human EAC cell lines and the differential susceptibility of these EAC cell lines to single agents or combinations of foretinib, a multi-kinase MET inhibitor, with HER2 targeted agent lapatinib. We evaluated the levels of phosphorylation status of MET and HER2 proteins using western blot in EAC cell lines. Foretinib and lapatinib, as single agent or in combination were tested for effect on cell growth as detected by WST-1 assay and on cell apoptosis as detected by western blot of cleavage of caspase 3 and poly ADP ribose polymerase (PARP). In addition, we explored the antitumor efficacy with survival advantage following foretinib and lapatinib mono and combination therapies for two weeks in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC. Results: The OE33 EAC cell line with phosphorylation of both MET and HER2, demonstrated reduced sensitivity to foretinib and lapatinib when used as a single agent. The co-administration of foretinib and lapatinib effectively inhibited both MET and HER2 phosphorylation, synergistically inhibited cell growth and induced apoptosis, overcoming single agent resistance. In the OE19 EAC cell line with only HER2 phosphorylation and the ESO51 EAC cell line with only MET phosphorylation, profound cell growth inhibition with induction of apoptosis was observed in response to single agent foretinib and lapatinib, respectively, with lack of enhanced growth inhibition when the two drugs were combined. Foretinib in combination with lapatinib treatment resulted in significantly higher antitumor efficacy and survival benefit compared with foretinib or lapatinib treatment alone. In subcutaneous xenografts using OE33 cells, average net tumor growth after two weeks in different therapy groups was 247.83 mm3 in control, 216.71 mm3 after foretinib (p=0.49), 239.68 mm3 after lapatinib (p=0.74), and 108.06 mm3 after foretinib plus lapatinib (p=0.0011). In the OE33 survival model there was a significant increase in median animal survival after two weeks foretinib plus lapatinib treatment (71 days) compared to control (60 days, p=0.0021), to foretinib therapy (63 days, p=0.0019) or to lapatinib (61 days, p=0.0019) therapy. Conclusion: These data suggest that combination therapy with foretinib and lapatinib should be tested as a treatment option for HER2 positive patients with MET-overexpressing EAC. Therefore, this combination therapy could be a novel treatment strategy for EAC with MET and HER co-activation. Citation Format: Md Sazzad Hassan, Fiona Williams, Niranjan Awasthi, Margaret A. Schwarz, Roderich E. Schwarz, Urs von Holzen. Synergistic effects of foretinib with lapatinib in MET and HER2 co-activated experimental esophageal adenocarcinoma [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 4826.

Abstract 2040: Superior therapeutic efficacy of nanoparticle albumin-bound paclitaxel over cremophor-bound paclitaxel in experimental esophageal adenocarcinoma

Introduction: Esophageal adenocarcinoma (EAC) has become the dominant type of esophageal cancer in United States. EAC is the fastest growing cancer in the western world and the overall 5 year survival rate of EAC is below 20 percent. Most patients with EAC present with locally advanced or widespread metastatic disease, where current treatment is largely ineffective. Prognosis for EAC patients remains poor even with combination therapies due to high resistance to chemotherapy. Therefore, new therapeutic approaches are urgently needed. Paclitaxel (PTX) has been used in combination with carboplatin (CP) as a standard combination therapy for advanced EAC. PTX required emulsification with solvents to allow intravenous administration which has resulted in hypersensitivity reactions and potentially dramatic side effects in patients. Nanoparticle albumin-bound (nab) PTX is an albumin-stabilized, cremophor-free and water soluble nanoparticle formulation of PTX. Nab-PTX is a novel microtubule-inhibitory cytotoxic agent and the potential role of nab-PTX has not been tested yet in experimental EAC. Methods: We explored the antiproliferative and antitumor efficacy with survival advantage following CP, PTX and nab-PTX as monotherapy and in combinations in in-vitro, and in murine subcutaneous xenograft and peritoneal metastatic survival models of human EAC. Results: Nab-PTX inhibited in-vitro cell proliferation with significantly lower IC50 (0.25 µM in OE19 and 49 nM in OE33) than that of PTX (0.74 µM in OE19 and 98 nM in OE33) and CP (5.21 µM in OE19 and 1.05 µM in OE33) in OE19 and OE33 EAC cell lines. Nab-PTX treatment resulted in significantly higher antitumour efficacy and survival benefit compared with PTX or CP treatment. After two-week nab-PTX, PTX, CP, nab-PTX+CP or PTX+CP treatments, the average in-vivo local tumor growth inhibition rate was 73, 60, 35, 81 and 68 percent respectively (p=0.025). Nab-PTX treatment increased expression of the mitotic-spindle associated phospho-stathmin, decreased expression of proliferative marker Ki-67 and enhanced apoptosis as confirmed by increased expression of cleaved-PARP and cleaved caspase-3. There was an increase in median animal survival after nab-PTX treatment (65 days) compared to controls (46 days, p=0.0023), PTX (57 days, p=0.0034) or to CP therapy (53 days, p=0.0034). Conclusion: In conclusion, the present study demonstrates that nab-PTX had stronger antiproliferative and antitumor activity in experimental EAC than the current standard chemotherapeutic agents. This strong antitumor activity supports the rationale for clinical evaluation of nab-PTX as promising microtubule-inhibitory agent in EAC. Citation Format: Md Sazzad Hassan, Niranjan Awasthi, Roderich E. Schwarz, Margaret A. Schwarz, Urs von Holzen. Superior therapeutic efficacy of nanoparticle albumin-bound paclitaxel over cremophor-bound paclitaxel in experimental esophageal adenocarcinoma [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 2040. doi:10.1158/1538-7445.AM2017-2040

Abstract 1258: Therapeutic potential of the cyclin- dependent kinase inhibitor on c-Myc overexpressing esophageal adenocarcinoma

Introduction: Esophageal adenocarcinoma (EAC) is now the fastest growing cancer in the western world and most EAC patients present with widespread metastatic disease, where current treatment is largely ineffective. Therefore, new therapeutic approaches are urgently needed. The transcription factor proto-oncogene c-Myc is a potent activator of tumorigenesis. Tumors with elevated c-Myc expression often exhibit highly aggressive phenotype. c-Myc amplification has been shown to be frequent in esophageal adenocarcinoma and has been implicated in Barrett9s carcinogenesis. Emerging data suggests that synthetic lethal interactions between c-Myc pathway activation and small molecules inhibition involved in cell cycle signaling can be therapeutically exploited to preferentially kill tumor cells. In this study, we therefore investigated whether exploiting a synthetic-lethal approach dependent on elevated c-Myc signaling is effective in treating esophageal cancer with a cyclin-dependent kinase (CDK) inhibitor flavopiridol. Methods: Western blot analysis was done to see the expression of c-Myc and apoptotic signaling pathways in a panel of nine esophageal cancer cell lines. c-Myc overexpression and knockdown were performed using both genetic and novel chemical approaches. Cell viability assays were performed in 96-well plates using the colorimetric WST-1 reagent. Esophageal cancer tumors growth was measured in xenograft and a novel peritoneal disseminated metastatic survival model of immunodeficient mice. Results: Western blot analysis revealed frequent overexpression of c-Myc in EAC cell lines. In this panel of esophageal cancer cell lines tested more than 70% of EAC cell lines showed overexpression of c-Myc. When we tested these cell lines for their ability to form xenograft tumor and peritoneal dissemination, c-Myc overexpression correlated with accelerated EAC tumor growth in xenograft and peritoneal disseminated metastatic survival model of NOD/SCID mice. The xenograft tumor growth rate and formation rate of peritoneal cancer after injection of 5 million cells were highest in OE19 EAC cell line which showed the highest c-Myc expression. In addition, median animal survival with peritoneal dissemination was lowest for OE19 (55 days) whereas OACM5.1 C EAC cell line which had the lowest c-Myc expression didn9t form any peritoneal tumor. EAC cell lines with elevated c-Myc expression are preferentially more sensitive to induction of apoptosis by CDK inhibitor flavopiridol compared to EAC cell lines with lower c-Myc expression. When we tested the role of c-Myc expression by upregulation/downregulation in this apoptotic effect we found that this effect is very much dependent on c-Myc expression. Conclusion: These results indicate that CDK inhibitor alone or in combination with other cytotoxic or targeted agents can be a potential therapy for c-Myc overexpressing EAC. Citation Format: Sazzad Hassan, Niranjan Awasthi, Margaret A. Schwarz, Roderich E. Schwarz, Urs V. Holzen. Therapeutic potential of the cyclin- dependent kinase inhibitor on c-Myc overexpressing esophageal adenocarcinoma. [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 1258.

Abstract 10: Effect of behavioral stress on therapeutic sensitivity of prostate cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Behavioral stress has been long implicated in cancer pathogenesis, yet mechanistic aspects of stress/tumor interactions are understudied. Several publications described effects of stress hormone epinephrine on tumor stroma components: vasculature and immune cells; however it was not clear whether epinephrine could influence cancer cells directly and if increase of epinephrine induced by behavioral stress could activate anti-apoptotic signaling in tumors and diminish efficacy of anti-cancer therapies. Here, we studied whether subjecting mice to behavioral stress increases resistance of PTEN-deficient prostate cancer to treatments with PI3K inhibitors. We examined effects of behavioral stress on C4-2Luc prostate cancer xenografts that express firefly luciferase and could be non-invasively monitored by luminescent imaging. In these tumor xenografts PI3K pathway is constitutively active due to the loss of PTEN expression. Injection of PI3K inhibitors into C4-2Luc xenografts induced apoptosis and substantially reduced luminescence. Subjecting mice to immobilization stress for 1 hour increased plasma epinephrine levels up to 10-15 nM, inhibited apoptosis and prevented reduction of luminescence in C4-2Luc xenografts treated with PI3K inhibitors. Injections of epinephrine had similar to stress effects. Conversely, the effect of either stress or epinephrine was completely blocked by beta2-adrenergic receptor (ADRB2) selective antagonist ICI118,551, implying that effects of stress/epinephrine are mediated via ADRB2. Elevated phosphorylation of BAD and CREB and inhibition of apoptosis was observed in xenograft tumors of mice subjected to immobilization stress. Effects of stress on BAD and CREB phosphorylation were completely blocked by ADRB2 selective antagonist ICI118,551. To test the role of protein kinase A (PKA) in anti-apoptotic signaling by stress/epinephrine in vivo we generated C4-2Luc cells that inducibly express chimera of PKA inhibitor peptide PKI and GFP (PKI-GFP). Induction of PKI-GFP expression in prostate cancer xenografts blocked stress/epinephrine-induced activation of PKA as judged by lack of CREB phosphorylation. It also prevented stress/epinephrine-induced activation of anti-apoptotic signals that phosphorylated BAD. Furthermore, C4-2Luc xenografts that inducibly express GFP-PKI were not protected from apoptosis by epinephrine or stress. In summary, we found that subjecting mice to behavioral stress resulted in increase of serum epinephrine level leading to activation of the ADRB2 and PKA pathway in prostate tumors. This ADRB2/PKA activation is necessary for in vivo BAD phosphorylation and protection of prostate tumors from apoptosis induced by PI3K inhibitors. Thus, elevated epinephrine levels could contribute to therapy resistance of prostate 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 10. doi:10.1158/1538-7445.AM2011-10

Abstract 183: Loss of Bad phosphorylation and Mcl-1 expression is necessary for rapid apoptosisinduced by combination of TGFα-PE and ZSTK474 in prostate cancer cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Advanced androgen-independent prostate cancer is notoriously resistant to conventional systemic therapies, and at the moment there is no effective protocol for hormone-refractory advanced prostate cancer affected patients. One of possible mechanisms of such resistance is activation of the anti-apoptotic signaling PI3K/Akt pathway that is constitutively active in the 60% of advanced prostate cancers. However, multiple inhibitors of PI3K are just well studied and some have just gone on to clinical trials, but unfortunately they showed limited efficacy against prostate cancer alone. Our recent experiments has shown remarkable synergy in inducing rapid and massive apoptosis when PI3K inhibitor ZSTK474 is used in combination with Pseudomonas aeruginosa exotoxin A fragment fused with Transforming Growth Factor Alpha (TGFα-PE38). Time lapse video microscopy of prostate cancer C4-2 cell line has shown the substantial cell death within 4-6 hours with combined administration, compared to limited cell death in single agents-treated cells. Analysis of PARP and cleaved-caspase 3 fragment by Western blotting confirmed that cell death occurs via apoptosis. Quantitation of caspase 3 and 7 activity with luminescent AFC-DEVD substrate confirmed synergy in apoptosis activation by combination of TGFα-PE38 and ZSTK474, while single agents even in higher concentration did not induce substantial apoptosis within 6 hours time period. Analysis of the mechanisms underlying this synergy has shown that PI3K inhibitor ZSTK474 triggers Bad dephosphorylation, while TGFα-PE38 reduces expression levels of Mcl-1. A slight increase of Bim protein was detected and related with ZSTK474 administration. No major variations were detected in the expression levels of pro- and anti-apoptotic proteins Bax, Bcl-2 and Bcl-XL. To address the role of Bad phosphorylation and Mcl-1 expression in apoptosis induction by PI3K inhibitor ZSTK474 and TGFα-PE38, we examined apoptosis in C4-2 cells that express phosphorylation-deficient mutant Bad (BAD2SA). Results indicated that the expression of BAD2SA sensitized C4-2 cells to apoptosis induced by TGFα-PE38. Experiments that address the role of Mcl-1 loss in apoptosis induced by TGFα-PE38 are ongoing. 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 183. doi:10.1158/1538-7445.AM2011-183