Ali Alaseem

Matrix Metalloproteinases: A challenging paradigm of cancer management

Matrix metalloproteinases (MMPs) are members of zinc-dependent endopeptidases implicated in a variety of physiological and pathological processes. Over the decades, MMPs have been studied for their role in cancer progression, migration, and metastasis. As a result, accumulated evidence of MMPs incriminating role has made them an attractive therapeutic target. Early generations of broad-spectrum MMP inhibitors exhibited potent inhibitory activities, which subsequently led to clinical trials. Unexpectedly, these trials failed to meet the desired goals, mainly due to the lack of efficacy, poor oral bioavailability, and toxicity. In this review, we discuss the regulatory role of MMPs in cancer progression, current strategies in targeting MMPs for cancer treatment including prodrug design and tumor imaging, and therapeutic value of MMPs as biomarkers in breast, lung, and prostate cancers.

Abstract 80: Analysis of the regulation of angiogenesis pathway by inhibiting MDM2 function in LNCaP-MST prostate cancer cells using PCR array

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Interfering with angiogenesis is a well-known strategy for cancer treatment due the fact that this process is essential for promoting cancer growth and metastatic spread by providing adequate supply of oxygen and nutrients to the tumor cells. The process of angiogenesis is highly regulated by both activator and inhibitor genes through their corresponding proteins. One of the suspected promoters of angiogenesis is MDM2 (Murine Double Minute 2 gene). The overexpression of MDM2 has been found in many cancer types and also correlated well with poor prognosis of certain cancers. In addition to some of the newly emerging mechanistic abilities, MDM2 is well known to regulate p53 levels and its functions by dual mechanisms, which plays a major role in many cellular processes such as cell cycle control and apoptosis. However, the role for MDM2 in regulating angiogenesis is slowly evolving which can explain the potential metastatic status of the cancer where there is no p53 mutation. In this connection, to further understand its role in tumor angiogenesis, the status of MMP9 (Matrix metallopeptidase 9 also called gelatinase B), THSB1 (Thrombospondin 1), TGB1 (Transforming growth factor-beta 1) were studied, by inhibiting MDM2. The ultimate goal of our study was to determine how the angiogenesis pathway is regulated by the overexpression of MDM2. To explore the possible mechanisms, LNCaP (prostate cancer cells), and LNCaP-MST (MDM2 transfected prostate cancer cells) cells were treated with Nutlin-3 (20 uM for 24 hours). Indeed LNCaP-MST cells are known to show 10 times higher expression of MDM2 compared to LNCaP cells. Therefore, Nutlin-3 treatment was expected to inhibit the interaction between p53 and MDM2 in these cells and thereby impact the expression of angiogenesis pathway related genes. When we analyzed the gene expression profile using a PCR array that was designed to explore the above mentioned pathway we were able to confirm that MDM2 overexpression could positively regulate MMP9, THSB1, and TGB1 levels in a highly significant manner. Interestingly, the genes expression profile that was found to be elevated in LNCaP-MST cells was reversed after Nutlin-3 treatment confirming that some of these angiogenesis related genes are positively regulated by MDM2 overexpression. In Conclusion, we are proposing that targeting MDM2 can effectively control the pro-angiogenesis mechanisms that can be found activated in MDM2 positive cancer types. We are conducting further studies to fully unravel the role of MDM2 in promoting tumor angiogenesis and metastatic ability (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale is gratefully acknowledged). Citation Format: Ali Alaseem, Thiagarajan Venkatesan, Khalid Alhazzani, Appu Rathinavelu. Analysis of the regulation of angiogenesis pathway by inhibiting MDM2 function in LNCaP-MST prostate cancer cells using PCR array. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 80. doi:10.1158/1538-7445.AM2015-80

MDM2 Overexpression Modulates the Angiogenesis-Related Gene Expression Profile of Prostate Cancer Cells

The Murine Double Minute 2 (MDM2) amplification or overexpression has been found in many tumors with high metastatic and angiogenic ability. Our experiments were designed to explore the impact of MDM2 overexpression, specifically on the levels of angiogenesis-related genes, which can also play a major role in tumor propagation and increase its metastatic potential. In the present study, we have used the human angiogenesis RT2 profiler PCR array to compare the gene expression profile between LNCaP and LNCaP-MST (MDM2 transfected) prostate cancer cells, along with LNCaP-MST cells treated with Nutlin-3, an MDM2 specific inhibitor. As a result of the overexpression of MDM2 gene in LNCaP-MST (10.3-fold), Thrombospondin 1 (THBS1), Tumor necrosis factor alpha (TNF-α) and Matrix metallopeptidase 9 (MMP9) were also found to be significantly up-regulated while genes such as Epiregulin (EREG), Tissue inhibitor of metalloproteinases 1 (TIMP1) were down-regulated. Also, we determined the total MMP activity and MMP9 expression in LNCaP, LNCaP-MST and SJSA-1 cells. Our results indicated that MDM2 level is positively correlated with MMP activity and MMP9 secretion. Our findings offer strong supporting evidence that MDM2 can impact growth and metastatic potential of cancer cells through tilting the balance towards pro-angiogenic mechanisms.

Focal Adhesion Kinase in Ovarian Cancer: A Potential Therapeutic Target for Platinum and Taxane-Resistant Tumors

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase, which is an essential player in regulating cell migration, invasion, adhesion, proliferation, and survival. Its overexpression and activation have been identified in sixty-eight percent of epithelial ovarian cancer patients and this is significantly associated with higher tumor stage, metastasis, and shorter overall survival of these patients. Most recently, a new role has emerged for FAK in promoting resistance to taxane and platinum-based therapy in ovarian and other cancers. The development of resistance is a complex network of molecular processes that make the identification of a targetable biomarker in platinum and taxane-resistant ovarian cancer a major challenge. FAK overexpression upregulates ALDH and XIAP activity in platinum-resistant and increases CD44, YB1, and MDR-1 activity in taxaneresistant tumors. FAK is therefore now emerging as a prognostically significant candidate in this regard, with mounting evidence from recent successes in preclinical and clinical trials using small molecule FAK inhibitors. This review will summarize the significance and function of FAK in ovarian cancer, and its emerging role in chemotherapeutic resistance. We will discuss the current status of FAK inhibitors in ovarian cancers, their therapeutic competencies and limitations, and further propose that the combination of FAK inhibitors with platinum and taxane-based therapies could be an efficacious approach in chemotherapeutic resistant disease.

Abstract 309: Effect of histone deacetylase (HDAC) inhibitor on gene expression in MDM2 transfected prostate cancer cells

Deacetylation of histone gives a tag for epigenetic repression and plays an important role in transcriptional regulation, cell cycle progression, and developmental events. HDACs catalyse the removal of the acetyl moiety from the lysine residues of proteins including the core nucleosomal histones. Through removal of critical acetyl groups from histones, HDACs can create a chromatin conformation that can prevent the transcription of genes that encode for proteins involved in cell cycle regulation. Thus together with histone acetyltransferases (HATs), HDACs regulate the level of acetylation and alter multitude of cellular functions and their characteristics. Several alterations of HDAC and HAT levels and activities have been found to be enacted by translocation, amplification, overexpression, or mutation of the relevant genes in a variety of cancers. In many cancer cell lines, overexpression or activation of the HDAC enzymes result in histone hypo-acetylation and consequent promotion of pro-cancerous mechanisms. Therefore, HDAC inhibitors represent a potential new class of antitumor agents with cytotoxic activity and the ability to regulate gene expression in tumor cells. In this study we evaluated the effects of Vorinostat (suberoylanilide hydroxamic acid), which is a potent inhibitor of HDAC activity, on cell cycle regulation in MDM2 (mouse double minute 2 homolog) overexpressing cells. MDM2 amplification or overexpression is found in many tumors that eventually lead to the inactivation of the cell cycle control and loss of pro-apoptotic functions through both p53 dependent and independent mechanisms. The PCR array, qRT-PCR, and western blot analysis of MDM2 overexpressing prostate cancer cells (LNCaP-MST), after treating with Nutlin-3 (20 µm) and 17-AAG (10 µm), was able to trigger p21 expression and down-regulation of BIRC5 (Baculoviral IAP Repeat Containing 5). Similarly, when we treated the MDM2 transfected LNCaP-MST cells with vorinostat (7.5 µm for 24 hrs), some of the above mentioned changes, similar to Nutlin-3 treatment, were observed. As a result of HDAC inhibition the mRNA levels of p21, p53 and TIMP-1 were significantly elevated, while the levels of BIRC5 was significantly down-regulated. Thus, treatment of MDM2 overexpressing cell lines with HDAC inhibitor resulted in activation of p21 and consequent decrease in cell proliferation due to resumption of cell cycle arrest. Our results with LNCaP-MST cells offer convincing evidence to suggest that the inhibition of HDAC can control cell proliferative signals in MDM2 overexpressing prostate cancer cells. (The generous support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). Citation Format: Thiagarajan Venkatesan, Ali Alaseem, Khalid Alhazzani, Thanigaivelan Kanagasabai, Appu Rathinavelu. Effect of histone deacetylase (HDAC) inhibitor on gene expression in MDM2 transfected prostate cancer cells [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 309. doi:10.1158/1538-7445.AM2017-309

Abstract 1844: MDM2 overexpression promotes angiogenesis and metastasis through dysregulation of MMP-9 and TIMP-1 pathway

MDM2 is a multifunctional oncogene that has been associated with highly prevalent and aggressive cancers such as colon and prostate. MDM2 promotes a negative regulation of p53 activities and thus can prevent several of p53 protective effects including DNA repair, cell cycle control and apoptosis. Several pharmacological blockers that target the N-terminal domain of MDM2 leading to a disruption of MDM2-p53 interaction are being developed. For instance, nutlin-3 is the prototype of this family and has been shown to confine the tumor growth in many cancer types. Recently, it has been confirmed that MDM2 also promotes metastasis and angiogenesis through activation of number of mediators such VEGF and HIF-1α. However, mounting evidence suggests that MDM2 has p53 independent functions. Therefore, understanding the mechanisms by which MDM2 promotes the metastatic ability of tumors might provide an invaluable therapeutic target and a potential prognostic biomarker. Particularly, MMP-9/TIMP-1 dysregulation has been known to be associated with cancer progression and metastasis. This system is involved in the digestion of extracellular matrix and thus a release of several sequestered cytokines and growth factors such as TGFβ1. Among the release cytokines, TGFβ1 has been proven to be involved in promoting the MMP-9 expression and consequent metastatic events. Therefore, we hypothized that MDM2 promotes angiogenesis and metastasis through dysregulation of TIMP-1/MMP-9 pathway. Hence, the goal of our study was to evaluate the influence of MDM2 on tumor angiogenesis and metastatic ability through dysregulation of TIMP-1/MMP-9 pathway. To test our hypothesis, LNCaP prostate cancer and MDM2-transfected LNCaP-MST cell lines were used. On the other hand, nutlin-3 at the dose of 20 μM for 24 hours were used to examine the influence of MDM2 in regulating MMP-9/TIMP-1 in LNCaP and LNCaP-MST cells. Utilizing qPCR and western blotting techniques, we obtained data which suggest that MDM2 positively regulates the expression of MMP-9 and negatively regulates TIMP-1. We observed that MMP-9 overexpressed in LNCaP-MST, and this overexpression was reversed after nutlin-3 treatment in LNCaP-MST by 0.40 fold. Conversely, the results showed a complete abolishment of TIMP-1 protein level in LNCaP-MST, and nutlin-3 does not seem to impact TIMP-1 level. The data suggest that MDM2 positively impacts MMP-9 in a p53 dependent fashion as opposed to TIMP-1, which is negatively regulated by MDM2 independent of p53. In the same connection, TGFβ1 overexpression was reversed by 0.51 fold in LNCaP-MST after nutlin-3 treatment as shown by the qPCR results. Altogether, this project would provide a significant insight into understanding cancer metastasis processes with respect to MDM2 activity (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale is gratefully acknowledged). Citation Format: Ali Alaseem, Thiagarajan Venkatesan, Priya Dondapati, Saad Alobid, Appu Rathinavelu. MDM2 overexpression promotes angiogenesis and metastasis through dysregulation of MMP-9 and TIMP-1 pathway. [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 1844.

Abstract 1839: Analysis of cell cycle-related gene expressions in MDM2-transfected LNCaP-MST cells after inhibition of HSP90

MDM2 gene is a cellular proto-oncogene amplified in 25-40% of all human cancers and regulates cell proliferation, senescence, and apoptosis through targeting p53. It is widely speculated that MDM2 protein is not only responsible for tumorigenesis via p53 inactivation, but also for increasing the metastatic ability of originally non-metastatic tumor cells. HSP90 is a molecular chaperone that regulates the maturation, activation and stability of critical signaling proteins that drive the development and progression of prostate cancer. 17-Allyamino-17-demethoxygeldanamycin (17-AAG) is an inhibitor of HSP90, which causes proteasomal degradation of client proteins such as AR, HER2, Akt etc., leading to cell cycle arrest and substantial growth inhibition. HSP90 is also believed to be involved in the stabilization of MDM2 and therefore, inhibition of HSP90 is expected to accelerate degradation of MDM2 and disrupt its function. Hence, our study was designed to determine the expression profile of the genes in the cell cycle pathway following treatment of MDM2 overexpressing LNCaP-MST prostate cancer cells with 17-AAG (10 μM at 24 hrs). In the present study, Human cell cycle PCR array was used to examine the alterations in gene expression pattern following exposure to the 17-AAG in MDM2 overexpressed prostate cancer cells. In our experiments we observed that the genes impacted by MDM2 overexpression were reversed by HSP90 inhibition. The expression of genes such as CDC25A, CDC25C, AURKB and Survivin levels were significantly down-regulated after 17-AAG treatment, while p21 and GADD45A were up-regulated compared to the control LNCaP-MST. A heat map and scatterplot analysis clearly confirmed the alterations in the expression levels of these genes following 17-AAG treatment. It is expected that more thorough understanding of the consequences of HSP90 blockade, particularly those that are independent of its role as a regulator of p53, will reveal its therapeutic significance. Our results offer convincing evidence to suggest that the inhibition of HSP90 can induce cell cycle arrest in LNCaP-MST cells. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). Citation Format: Thiagarajan Venkatesan, Ali Alaseem, Khalid Alhazzani, Priya Dondapati, Saad Alobid, Appu Rathinavelu. Analysis of cell cycle-related gene expressions in MDM2-transfected LNCaP-MST cells after inhibition of HSP90. [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 1839.

Abstract 81: Effect of HSP90 inhibition on the gene expression profile of MDM2 transfected LNCaP-MST prostate cancer cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The heat shock protein 90 (HSP90) is a major molecular chaperone of the cell that appears to be a master regulator of the stability and activity of multiple oncoproteins such as HER2, Akt, Bcr-Abl, c-Kit, EGFR and BRAF. Recent approaches have revealed that most of the target proteins of HSP90 are protein kinases or transcription factors which play important roles in the cellular carcinogenesis. The HSP90 also plays an important role in the regulation of cell cycle, proliferation, vascularization, invasion and metastasis of cancer cells, as well as in maintaining the stability and functions of several carcinogenic proteins involved in altering the signal transduction pathways of apoptosis. The 17-allylamino-17-demethoxygeldanamycin (17-AAG) is a well known inhibitor of HSP90 and it was speculated that, this inhibitor may have particular value in the antitumor and angiostatic therapies. Hence, we were trying to determine the expression profile of the genes in the angiogenesis pathway following treatment of MDM2 overexpressing LNCaP-MST prostate cancer cells with 17-AAG. In the LNCaP-MST cells, the expression of genes such as TNF-α, MMP9 and TGFB1 were found to be significantly up-regulated compared to the LNCaP cells. Hence, MDM2 overexpression was suspected as the primary cause of these increases. However, after treating the LNCaP-MST cells with 10 μm 17-AAG for 24 hrs, several of these genes were significantly altered compared to the untreated LNCaP-MST cells. In particular, the 17-AAG treatment significantly down-regulated the expression of TNF-α, TGFB1, ID1, IL8 and F3 which are involved in the regulation of angiogenesis and tumor growth. The heat map and scatterplot analysis clearly confirmed the alterations in the expression levels of these genes following 17-AAG treatment. Cluster analysis further indicated that the gene expression patterns are positively correlated with the anti-angiogenic effects of 17-AAG treatment in LNCaP-MST cells compared to untreated cells. Our results offer convincing evidence to suggest that the inhibition of HSP90 can control both pro-angiogenic and cell proliferative signals in LNCaP-MST cells. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). Citation Format: Thiagarajan Venkatesan, Ali Alaseem, Khalid Alhazzani, Appu Rathinavelu. Effect of HSP90 inhibition on the gene expression profile of MDM2 transfected LNCaP-MST prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 81. doi:10.1158/1538-7445.AM2015-81

Abstract 82: Analysis of the role of MDM2 in regulation of cell cycle arrest through p21 pathways in LNCaP-MST cells using PCR array

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Cell cycle checkpoints are imperative for assuring genomic fidelity and integrity during cell division. In normal cells, the cell cycle arrest occurs in response to nuclear DNA damage via activation and inactivation of a series of cell cycle regulatory molecules that can impact the transition from G1 to S phase and G2 to M phase. However, in cancerous cells, this process is disrupted resulting in uncontrolled cell proliferation and resistance to apoptosis. Therefore, cell cycle regulatory molecules are accepted as suitable targets for drug development and cancer treatment. It has been well known that overexpression of MDM2, a negative regulator of p53, will eventually lead to the inactivation of cell cycle control and loss of apoptotic ability in many tumors. In this study, we aimed to investigate the effectiveness of MDM2 inhibition in mediating cell cycle arrest through p53 dependent pathways by using the MDM2 transfected prostate cancer cells (LNCaP-MST). During our experiments the LNCaP-MST cells were treated for 24 hrs with 20 uM Nutlin-3, a small molecule inhibitor of MDM2/p53 interaction. The impact of Nutlin-3 treatment on the gene expression profile of LNCaP-MST cells was established by using the cell cycle pathway specific PCR array. Our study clearly demonstrates a significant increase of p21 gene expression after Nutlin-3 treatment, which indicates a possible restoration and release of the transcriptional activity of p53 in LNCaP-MST cells. In addition to the elevation of p21, a significant increase in the expression of Cyclin-Dependent Kinase 4 Inhibitor B (CDKN2B) along with multiple folds increase in the expression of Growth Arrest and DNA Damage 45 (GADD45A) genes were also found. These results clearly point towards efficient initiation of cell cycle arrest mechanisms in LNCaP-MST cells, possibly through p21 and CDKN2B mediated inhibition of the CDKs (cyclin dependent kinases). In addition, it appears that MDM2 inhibition may effectively block both G1 to S and G2 to M transition and cause DNA damage through apoptosis that could lead to the death of LNCaP-MST cells. Activation of the above outlined mechanisms following MDM2 inhibition is quite evident due to increased expression of GADD45A and decreased levels of anti-apoptotic B-cell lymphoma 2 (Bcl-2) genes. Our results offer convincing evidence towards the effectiveness of MDM2 inhibition in causing cell cycle arrest and apoptosis during cancer treatment. (This research was supported by the generous funds provided by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida) Citation Format: Khalid Alhazzani, Ali Alaseem, Thiagarajan Venkatesan, Appu Rathinavelu. Analysis of the role of MDM2 in regulation of cell cycle arrest through p21 pathways in LNCaP-MST cells using PCR array. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 82. doi:10.1158/1538-7445.AM2015-82