Ahmed S. Sultan

Stat5 promotes homotypic adhesion and inhibits invasive characteristics of human breast cancer cells

Signal transducer and activator of transcription-5 (Stat5) mediates prolactin (PRL)-induced differentiation and growth of breast epithelial cells. We have recently identified active Stat5 as a tumor marker of favorable prognosis in human breast cancer, and determined that Stat5 activation is lost during metastatic progression. Here we provide novel evidence for an invasion-suppressive role of Stat5 in human breast cancer. Activation of Stat5 by PRL in human breast cancer lines was associated with increased surface levels of the invasion-suppressive adhesion molecule E-cadherin in vitro and in xenotransplant tumors in vivo. Inducible E-cadherin was blocked by dominant-negative (Dn) Stat5 or Dn-Jak2, but not by Dn-Stat3. Further experimental data indicated a role of Stat5 as a coordinate regulator of additional invasion-related characteristics of human breast cancer cells, including cell surface association of β-catenin, homotypic cell clustering, invasion through Matrigel, cell migration, and matrix metalloproteinase activity. A role of Stat5 as a suppressor of breast cancer invasion and metastatic progression provides a biological mechanism to explain the favorable prognosis associated with active Stat5 in human breast cancer.

Co‐overexpression of Janus kinase 2 and signal transducer and activator of transcription 5a promotes differentiation of mammary cancer cells through reversal of epithelial–mesenchymal transition

Signal transducer and activator of transcription (Stat) 5 appears to play a vital role in prolactin (PRL)‐induced cell differentiation and normal mammary gland development. We previously showed that PRL‐activated Stat5a induced expression of E‐cadherin–β‐catenin complex in vitro and in xenotransplant tumors in vivo led to inhibition of breast cancer invasion. In the present study, we show that human breast cancer cells co‐overexpressing Stat5a and its tyrosine kinase (Jak) 2 cultured in three‐dimensional (3D) Matrigel culture demonstrate changes consistent with induction of mesenchymal–epithelial redifferentiation. Jak2 and Stat5a‐co‐overexpressing cells treated with cocktail (PRL, dexamethasone, and insulin), effectively reverse epithelial–mesenchymal transition by stimulating 3D organoids more reminiscent of the acinar growth of normal mammary epithelial cells, compared with cells overexpressing only Stat5a or Jak2. In contrast, dominant‐negative dominant‐negative‐Stat5 blocks 3D organoid formation, causing cells to grow in layers instead. Hyperactivation of Jak2 and Stat5a in T‐47D cells induces alveolar‐like structures, mamospheres, with marked lumen formation through central apoptosis and restores a polarized epithelial phenotype. However, Jak2 and Stat5a overexpression in BT‐20 cells induces partially differentiated 3D organoids with no central lumen, but effectively re‐expresses estrogen receptor α. Jak2 and Stat5a‐induced 3D differentiated organoids are accompanied by increased expression of E‐cadherin, zonula occludens‐1, and cytokeratins 8 and 18, and decreased levels of vimentin and Snail, indicating a shift from a mesenchymal phenotype toward an epithelial phenotype. Collectively, Jak2 and Stat5a co‐overexpression cooperatively reverses epithelial–mesenchymal transition and promotes differentiation in human breast cancer cells, which may provide a mechanism to explain the invasive suppressor role of PRL‐activated Stat5a in mammary cancer cells. (Cancer Sci 2008; 99: 272–279)

Ultrahigh density microarrays of solid samples

We present a sectioning and bonding technology to make ultrahigh density microarrays of solid samples, cutting edge matrix assembly (CEMA). Maximized array density is achieved by a scaffold-free, self-supporting construction with rectangular array features that are incrementally scalable. This platform technology facilitates arrays of >10,000 tissue features on a standard glass slide, inclusion of unique sample identifiers for improved manual or automated tracking, and oriented arraying of stratified or polarized samples.

Histone deacetylase inhibitors sensitize lung cancer cells to hyperthermia: involvement of Ku70/SirT-1 in thermo-protection.

This study describes the sensitization mechanism to thermal stress by histone deacetylase inhibitors (HDACIs) in lung cancer cells and shows that Ku70, based on its acetylation status, mediates the protection of lung cancer from hyperthermia (42.5°C, 1-6 hrs). Ku70 regulates apoptosis by sequestering pro-apoptotic Bax. However, its role in thermal stress is not fully understood. The findings showed that, pre-treating lung cancer cells with HDACIs, nicotinamide (NM) or Trichostatin A (TsA) or both significantly enhanced hyperthermia-induced Bax-dependent apoptosis in PC-10 cells. We found that hyperthermia induces SirT-1, Sirtuin, upregulation but not HDAC6 or SirT-3, therefore transfection with dominant negative SirT-1 (Y/H) also eliminated the protection and resulted in more cell death by hyperthermia, in H1299 cells through Bax activation. Hyperthermia alone primed lung cancer cells to apoptosis without prominent death. After hyperthermia Bax was upregulated, Bcl-2 was downregulated, the Bax/Bcl-2 ratio was inversed and Bax/Bcl-2 heterodimer was dissociated. Although hyperthermia did not affect total Ku70 expression level, it stimulated Ku70 deacetylation, which in turn could bind more Bax in the PC-10 cells. These findings suggest an escape mechanism from hyperthermia-induced Bax activation. To verify the role of Ku70 in this protection mechanism, Ku70 was silenced by siRNA. Ku70 silencing significantly sensitized the lung cancer cells to hyperthermia. The Ku70 KD cells underwent cytotoxic G1 arrest and caspase-dependant apoptosis when compared to scrambled transfectants which showed only G2/M cytostatic arrest in the cell lines investigated, suggesting an additional cell cycle-dependent, novel, role of Ku70 in protection from hyperthermia. Taken together, our data show a Ku70-dependent protection mechanism from hyperthermia. Targeting Ku70 and/or its acetylation during hyperthermia may represent a promising therapeutic approach for lung cancer.

Abstract 4440: New predictive and preventive biomarkers for preclinical hepatocellular carcinoma in Egyptian population by detection of serum miRNAs

The hepatocellular carcinoma (HCC) is one of the most common malignant tumors and carries a poor survival rate. Prognosis, survival and management of patients at risk for developing HCC remain challenging in Egypt and worldwide. Furthermore, poor prognosis of patients with symptomatic (HCC) diagnosed clinically at advanced stages suggests an urgent need for new biomarkers detection that can be used for pre-clinical screening for early detection of premalignant lesions and tumors in high risk to hepatitis C infection. In the present study we used novel formulated anticancer compound(s), combined with or without Sorafenib treatment to exert their antiproliferative effects by eliciting concomitant expression of known or novel biomarkers, including different serum miRNAs, which can be quantitatively analyzed for differences in the levels of metabolites and proteins in the blood and liver tissues of treated HCC xenografts and liver cirrhosis animal models. In addition, circulating miRNAs levels were measured as promising markers in 12 month interval-serum samples that were collected from normal, chronic hepatitis C virus infected patient cohorts at the pre-malignant or pre-clinical stages and liver cirrhotic patients that did not develop cancer. Different serum miRNAs, including miR-21, miR-142, let-7a, let-7b, miR-429 and miR-34a were detected in pre-clinical HCC patients and have the potential to screen for hepatitis C virus infected patients at high risk to develop HCC 12 months after miRNAs detection. Our data of detected serum miRNAs from animal models and patient samples can be used to confirm and correlate histological and histochemical results at various time points with serum or imaging biomarkers, which may have a great promise for the prediction and prevention of HCC in high risk Egyptian populations. Citation Format: Sara Salama Ghazy, Amira S. Fyala, Ahmed S. Sultan. New predictive and preventive biomarkers for preclinical hepatocellular carcinoma in Egyptian population by detection of serum miRNAs [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 4440. doi:10.1158/1538-7445.AM2017-4440

Abstract 318: Trigonella foenum (fenugreek) inhibited cancer stem cell properties and increased sensitivity to Doxorubicin resistant ZR-75-1 human breast cancer cell line by targeting mutant p53 and Notch-4

Despite advances in detection and therapies, breast cancer is the most common malignant disease in women worldwide. Doxorubicin is one of the effective agent for breast cancer treatment, but the resistance to it is representing a major obstacle for breast cancer treatment. Herein, we established a breast cancer cell line that showed a significant resistance to doxorubicin at clinically relevant concentrations. The epithelial breast cancer cell line ZR-75-1 was sequentially treated with doxorubicin for several treatment cycles. In consequence, we obtained chemoresistant cells displaying a mesenchymal-like phenotype, ZR-75-1-DR. The ZR-75-1-DR cells showed an increase in breast cancer stem cells (BCSC) activity, mutant p53 but not wild type, NOTCH-4 and microRNAs, small noncoding RNAs, expression compared to parental cells, suggesting their expression may contribute to the evolvement of BCSCs. Understanding the early molecular mechanisms of cross talk among mutant p53, Notch-4 and microRNAs in resistant to doxorubicin is crucial to provide better therapeutic strategies for breast cancer progression. Recently, we showed that Trigonella foenum (Fenugreek) extract (FCE), a traditional herbal plant with anti-tumor activity, induced apoptosis in HepG2 cells mediated by up-regulation of wild P53. To investigate the possible effect of FCE in the expression levels of mutant p53, Notch-4 and miRNAs in ZR-75-1-DR and parental cell lines, cells were pre-treated with or without FCE and/or doxorubicin for different time intervals. miRNAs were quantified using qRT-PCR and p53, Notch-4 expression and BCSC phenotype were evaluated by western blot analysis and immunohistochemistry. Our results revealed that FCE treatment showed a significant decrease in cell viability, clonogenicity and invasion capacity with a decrease in expression of mutant p53 and Notch-4 in ZR-75-1-DR compared to untreated cells. Furthermore, the inhibition of mutant p53 and Notch-4 expression was associated with a significant decrease in miR-21 and miR-142 with an increase in expression of let-7a and miR-34a. Furthermore, ZR-75-1-DR pre-treated with FCE showed an increase in chemosensitivity to doxorubicin at low doses with reduction in expression of stemness factor, ALDH1, compared to the parental and doxorubicin treated cells. In vivo, knockdown both mutant p53 and NOTCH-4 showed a significant regression in tumor size of ZR-75-1-DR xenografts compared to those of parental cells and control lentivirus after combined treatment with FCE and doxorubicin, but not with doxorubicin alone. All in all, our data introduced FCE as a promising non-toxic herbal, which has fourteen bioactive compounds and therapeutic potential to reduced cancer stem cell properties and increased sensitivity to doxorubicin treatment, by directly targeting mutant p53, NOTCH4 and microRNAs. Citation Format: Ahmed S. Sultan, Amira S. Fayala, Marwa Elkamel, Reem Bakir, Fatma Foad. Trigonella foenum (fenugreek) inhibited cancer stem cell properties and increased sensitivity to Doxorubicin resistant ZR-75-1 human breast cancer cell line by targeting mutant p53 and Notch-4. [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 318.

Alternative suppression strategy for human breast cancer invasion and metastasis: a potential for a new differentiation therapy.

Abstract #2059 Poor prognosis of human breast cancer is associated with a high potential of cancer cells invasion and metastasis. Epithelial-mesenchymal transition (EMT) is a key event in the tumor invasion process. We have recently uncovered novel evidence that signal transducers and activators of transcription 5a (Stat5a) and its Janus tyrosine kinase, (Jak2), suppress invasion and promote human breast cancer cells differentiation through inhibition of epithelial-to-mesenchymal dedifferentiation.
 However, the precise mechanism of Jak2/Stat5a role in human breast cancer is still under investigation.
 In this study, we have further critically taken advantage of the combination of Jak2/Stat5a genes expression, HDAC inhibitors, and Mistletoe (Viscum album) plant extracts as an alternative way to suppress breast cancer progression by reversing the mesenchymal phenotype, and restoring homotypic adhesion, and in turn induce tumor differentiation in vitro and in vivo. We have expanded our investigation to involve a highly invasive breast cancer cell line, MDA-231, in addition to BT-20 and T-47D human breast cancer cells that display different phenotypic and differentiation patterns ranging from poor to high differentiation. Cells were pretreated with or without different HDAC inhibitors, and/or Mistletoe (Viscum album) plant extracts, and then either mock infected or infected with adenovirus carrying Wild type (Wt-Jak2), Wt-Stat5a, Dominant negative (Dn-Stat5), or a combination of Wt-Jak2 and Wt-Stat5a.
 Using three-dimensional (3D) Matrigel model system in vitro, the pretreatment with HDAC inhibitors and/or Mistletoe (Viscum album) plant extracts synergized with co-expression of Wt-Jak2/Stat5a genes to promote epithelial differentiation by reversing the mesenchymal phenotype morphology, inducing homotypic adhesion, and inhibiting cell motility and invasiveness.
 We have also established a xenograft tumor model of MDA-231 cells in nude mice for in vivo studies. After 4 week, the growth of MDA-231 tumors that co-expressing Wt-Jak2/Stat5a was inhibited by 47.90% compared to the mock group (t=2.821, P Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2059.