Somya Shanmuganathan

Simultaneous inhibition of cell-cycle, proliferation, survival, metastatic pathways and induction of apoptosis in breast cancer cells by a phytochemical super-cocktail: genes that underpin its mode of action.

Traditional chemotherapy and radiotherapy for cancer treatment face serious challenges such as drug resistance and toxic side effects. Complementary / Alternative medicine is increasingly being practiced worldwide due to its safety beneficial therapeutic effects. We hypothesized that a super combination (SC) of known phytochemicals used at bioavailable levels could induce 100% killing of breast cancer (BC) cells without toxic effects on normal cells and that microarray analysis would identify potential genes for targeted therapy of BC. Mesenchymal Stems cells (MSC, control) and two BC cell lines were treated with six well established pro-apoptotic phytochemicals individually and in combination (super cocktail), at bioavailable levels. The compounds were ineffective individually. In combination, they significantly suppressed BC cell proliferation (>80%), inhibited migration and invasion, caused cell cycle arrest and induced apoptosis resulting in 100% cell death. However, there were no deleterious effects on MSC cells used as control. Furthermore, the SC down-regulated the expression of PCNA, Rb, CDK4, BcL-2, SVV, and CD44 (metastasis inducing stem cell factor) in the BC cell lines. Microarray analysis revealed several differentially expressed key genes (PCNA, Rb, CDK4, Bcl-2, SVV, P53 and CD44) underpinning SC-promoted BC cell death and motility. Four unique genes were highly up-regulated (ARC, GADD45B, MYLIP and CDKN1C). This investigation indicates the potential for development of a highly effective phytochemical combination for breast cancer chemoprevention / chemotherapy. The novel over-expressed genes hold the potential for development as markers to follow efficacy of therapy.

TGF-β2: A Novel Target of CD44-Promoted Breast Cancer Invasion

We have developed a tetracycline (tet)-off regulated expression of CD44s gene in the breast cancer (BC) cell line MCF-7 (B5 clone) and identified TGF-β2 (Transforming Growth Factor beta-2; 3 fold induction) as a potential CD44-downstream transcriptional target by microarray analysis. To further validate this finding, the same RNA samples, used for microarray analysis and their corresponding protein lysates, collected from the BC cell line MCF-7-B5, were examined for CD44 expression in the presence of HA. Our results showed that TGF-β2 mRNA levels were significantly elevated following the removal of tetracycline at 18, 24, and 48 h post-HA stimulation compared to the parental cells. Furthermore, the TGF-β2 precursor protein increased in a time-dependent pattern upon HA-stimulation and in the absence of tetracycline. More interestingly, inhibition of CD44 gene by RNAi method decreased TGF-β2 expression upon HA-stimulation, and subsequently inhibited BC cell invasion in vitro. In addition to identifying TGF-β2 as a target for HA/CD44 signaling, this data suggests that ATF/CREB might be a potential transcription factor linking HA/CD44 activation to TGF-β2 transcription and additional experiments are required for a better understanding of the molecular mechanisms underpinning the novel function of the CD44/ TGF-β2 signaling pathway in breast cancer metastasis.

Abstract P2-03-19: Discovery of the genes that underpin the transition to malignant phenotype of breast tumors in highly consanguineous region

Breast cancer (BC), a multifactorial and heterogeneous disease is a predominant women form of cancer worldwide affecting 22.9%. The rationale of this study is based on the following observations: 1) in Oman, a significantly increasing number of younger females (25-40 years) present to the clinic with advanced stage of BC; 2) in Oman, the rate of consanguinity is significantly high (52%); and 3) the transition from normal/begnin to malignant phenotype of breast tumor requires the involvement of a subset of specific genes. The long-term objective of this study is to identify and validate the subset of genes that are responsible for this malignant transformation using functional genomic studies, focus on this young age group of patients attending BC clinic (sporadic and familial). RNA samples were isolated from 40 Breast Tumors and 40 Normal tissues and analyzed by Microarray Gene Expression Profiling. Among a number of genes that were up and down regulated, BRIP1, HOXB3 and MAGED1 were identified as potential genes that might underpin the transition to the malignant phenotype; these genes were validated by RT-PCR using the same RNA samples that were examined by microarray. Pathway analysis was carried out to identify the major functional pathways connecting these genes. Ongoing sequencing of these genes using DNA extracted from the same samples will ultimately identify any genetic alteration that can affect the normal function of these genes. Functional validation assays aim to validate further the physiological relevance of these genes in tumor malignancy, and perhaps other novel genes specific to BC in the Omani population. Identification and validation of these genes will potentially pave the way towards the design of anti-cancer therapeutic strategies. Citation Format: Ishita Gupta, Allal Ouhtit, Somya Shanmuganathan, Hamad Al-Riyami. Discovery of the genes that underpin the transition to malignant phenotype of breast tumors in highly consanguineous region [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-19.

Abstract 1257: From chemoprevention strategy to identification of potential therapeutic targets and biomarkers for breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Over the years, several phyto-compounds have been extensively used in Complementary Alternative Medicine (CAM) studies, individually and often at higher doses to kill cancer cells. Based on the combination and synergism theory, we had previously demonstrated that a combination of Resveratrol and Indole-3-Carbinol synergized and killed a maximum number of Breast Cancer cells. In the present study, we have tested various combinations of 10 well known phytochemicals, used at bioavailable levels, for their effect on cell growth and proliferation of the MDA-MB-231; breast cancer (BC) cell line and MCF-10A; normal breast epithelium as control cell line. The results revealed a super combination of 7 phyto-compounds (7SC), that synergized and induced 100% clearance of the BC cells but did not affect the normal breast epithelial cells. Next, in order to understand the underlying molecular mechanisms of this ‘synergism’ effect, microarray analysis will be conducted on the mRNA collected from the 7SC treated and control cells at 12 and 24 hour time points. Ongoing in vivo experiments aim to evaluate the efficacy of the 7SC phyto-compound treatment in preventing tumor growth using xenograft mouse BC model, and further validate the functional relevance of these genes in BC cell growth and survival. The data supports our hypothesis that the 7SC could be used in CAM as a dietary supplement approach against BC, and further identify genes that have the potential to serve as biomarkers and gene candidate to guide the design of appropriate anti-BC therapeutic strategies. Note: This abstract was not presented at the meeting. Citation Format: Allal Ouhtit, Somya Shanmuganathan, Ishita Gupta, Hamad Al-Riyami, Madhwa Raj. From chemoprevention strategy to identification of potential therapeutic targets and biomarkers for breast cancer. [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 1257. doi:10.1158/1538-7445.AM2014-1257

Abstract 3302: CD146, a suppressor of breast cancer, is a novel target of CD44-signaling

More than 80% of cancer related deaths are caused by cancer cell metastasis, a crucial step in the onset of tumor development. Among the molecules involved in promoting cancer metastasis is the cell adhesion molecule CD44, whose role in promoting cancer cell motility and metastasis is well known. Despite this knowledge, the molecular mechanism through which CD44 promotes tumor development and cell metastasis is still unclear. CD146 (MUC 18) is another member of the cell adhesion molecule family, first identified in highly metastatic melanomas. The absence of CD146 in normal melanocytes and its high expression in melanomas suggests its tumor promoting actions. Despite the association between CD146 expression and development of melanoma, the expression patterns and the role of CD146 in normal and metastatic breast tissues are still controversial. In this paper we provided evidence clarifying some of these discrepancies by presenting CD146 as a negative downstream target for CD44. Finally, this study demonstrates a new role of CD44 in regulating neovascularization and in promoting cancer cell transmigration of blood vessels via regulation of its downstream target CD146. Citation Format: Mohamed E. Abdraboh, Hamad Al-Riyami, Yahya Al-Farsi, Allal Ouhtit, Andrew D. Hollenbach, Ishita Gupta, Somya Shanmuganathan, Madhwa Hg Raj. CD146, a suppressor of breast cancer, is a novel target of CD44-signaling. [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 3302. doi:10.1158/1538-7445.AM2014-3302

Abstract A72: TGF-β2 underpins CD44-promoted breast tumor cell invasion

Hyaluronan (HA) mediates communication between cancer cells and the environment via interactions with the cell surface receptor CD44. The long-term objective of this study is to increase our understanding of the mechanisms by which CD44-HA interaction promotes BC metastasis, and further identify and validate CD44-downstream transcriptional targets for anti-metastatic therapy. Here, we have developed a tetracycline (tet)-regulated expression of CD44 gene in the BC cell line MCF-7 (B5 clone) and identified TGF-beta2 (Transforming Growth Factor beta-2; 3 fold induction) as a potential CD44s-downstream transcriptional target by microarray analysis. The same RNA samples, used for microarray analysis and their corresponding protein lysates, were examined for CD44 expression by RT-PCR and western-Blot methods, respectively. TGF-beta2 mRNA levels were significantly elevated following the removal of tet at 18, 24, and 48 h post-HA stimulation compared to controls. Furthermore, the TGF-beta2 precursor protein increased in a time-dependent pattern upon HA-stimulation and in the absence of tet. Inhibition of CD44 gene by RNAi method decreased TGF-beta2 expression upon HA-stimulation and in the absence of tet. Our data put together strongly support the hypothesis that TGF-beta2 is a potential target of HA/CD44-downstream-signaling mediating BC cell invasion. Finally, CREB might be the transcription factor linking CD44 to TGF-beta2 transcription. Citation Format: Allal Ouhtit, Samineh Madani, Ishita Gupta, Somya Shanmuganathan, Hamad Al-Riyami, Yahya Al-Farsi. TGF-β2 underpins CD44-promoted breast tumor cell invasion. [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 A72.

Abstract A71: CD146 is a novel target of CD44-signaling mediating breast tumor invasion

Cancer cell metastasis is one of the most critical steps in tumor development and is responsible for more than 80% of cancer related deaths. The cell adhesion molecule CD44 promotes cancer cell motility and metastasis. Despite this knowledge, the molecular mechanism through which CD44 promotes tumor development and cell metastasis is still unclear. The CD146 (MUC 18), another member of the cell adhesion molecules family, first identified in highly metastatic melanomas. The absence of CD146 in normal melanocytes and its high expression in melanomas suggests its tumor promoting actions. However, CD146 appears to suppress breast tumor progression. Furthermore, despite the association between CD146 expression and development of melanoma, the expression patterns and the role of CD146 in normal and metastatic breast tissues are still controversial. In a pilot study, we have identified CD146 as a potential downstream target of CD44-signaling in the MDA-435 breast cancer cell line, using subtractive hybridization and northern blot analyses. In this paper we provide evidence that CD146 is a downstream target for CD44, in a way that CD146 expression is regulated according to tumor microenvironment. Finally, this study demonstrates a new role for CD44 in regulating neovascularization and promoting cancer cell transmigration of blood vessels via CD146. Citation Format: Allal Ouhtit, Mohamed Abdraboh, Andrew D. Hollenbach, Ishita Gupta, Somya Shanmuganathan, Hamad Al Riyami, Youssef Errami, Yahya Al Farsi, Madhwa HG Raj. CD146 is a novel target of CD44-signaling mediating breast tumor invasion. [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 A71.

Abstract 4384: Discovery of CD146/Latexin as a novel pathway suppressing breast tumor invasion.

CD146, also known as melanoma cell adhesion molecule or MUC18, was initially identified as a promoter of melanoma as well as prostate cancer progression and metastasis. Interestingly, however, CD146 appears to be a breast cancer (BC) tumor suppressor. The molecular mechanism of CD146 in cancer are still nascent and controversial. To address this discrepancy, we applied microarray gene expression profiling analysis, using a tetracycline (tet On)-inducible system of CD146 in the MDA-MB-231 BC cell line, and identified latexin (LXN) as a potential CD146-downstream signaling transcriptional target. LXN showed a 7 fold increase, which was confirmed in vitro by both time-course western blotting and RT-PCR analyses. In addition, NF-kappaB was identified as the molecular link between CD146 induction and LXN transactivation. More interestingly, we established the CD146-tet On inducible system in mouse BC xenograft model, and demonstrated that in vivo induction of CD146-inhibited breast tumor growth. Furthermore, breast tumor tissues from both Omani patients and patients from New Orleans area in Louisiana, were examined by immunohistochemistry for the expression of CD146 and LXN. Strikingly, the results revealed that the expression levels of both CD146 and its downstream target LXN showed parallel inhibition patterns in BC progression, with high expression in both normal and benign but low or no expression in malignant and metastatic breast tumor tissues. This study is the first to uncover a molecular link and provide evidence for a role of CD146/LXN-signaling in suppressing BC progression. Thus, reactivation of LXN through pharmacological manipulation holds particular promise for guiding the development of novel targeted-therapeutic strategies for BC. Citation Format: Allal Ouhtit, Zakariya Y. Abd Elmageed, Augusta Fernando, Rajiv Gaur, Ishita Gupta, Somya Shanmuganathan, Hamad Al-Riyami, Madhwa HG Raj. Discovery of CD146/Latexin as a novel pathway suppressing breast tumor invasion. [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 4384. doi:10.1158/1538-7445.AM2013-4384

Abstract A73: In vivo evidence of TIMPv as a potential target of CD146-signaling inhibiting breast tumor invasion

CD146, also known as melanoma cell adhesion molecule (Mel-CAM, MCAM) or MUC18, was initially identified as a marker of melanoma progression and a promoter of melanoma and prostate cancer (PC) metastasis. In contrast, CD146 appears to play a tumor suppression role in breast cancer (BC). Here, we applied microarray gene expression profiling analysis, using a tetracycline (tet On)-inducible system of CD146 in the MDA-MB-231 BC cell line, and we identified TIMPv (a variant of Tissue Inhibitor of Metalloproteinases), showing a 7 fold increase, as a potential CD146-downstream signaling transcriptional target; this was confirmed in vitro by both time-course western blotting and RT-PCR. Furthermore, breast tumor tissues from both Omani patients and patients from New Orleans area in Louisiana, were examined by immunohistochemistry for the expression of CD146 and TIMPv. Strikingly, the results revealed that the expression levels of both CD146 and its downstream target TIMPv showed parallel inhibition patterns in BC progression, with high expression in normal and benign but low or no expression in malignant and metastatic breast tumor tissue. These findings support the hypothesis that the expression of both, CD146 and its downstream target TIMPv, is lost during BC progression and metastasis. Ongoing studies in our laboratory aim to shed light on the molecular components linking CD146 to the activation of TIMPv transcription. Citation Format: Allal Ouhtit, Zakariya Y. Abd Elmageed, Sunil Bhat, Pravrutha Raman, Suad H. Al-Jardani, Ishita Gupta, Somya Shanmuganathan, Hamad Al-Riyami. In vivo evidence of TIMPv as a potential target of CD146-signaling inhibiting breast tumor invasion. [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 A73.

Abstract P6-04-16: TGF-b2, a novel target of CD44-promoted breast cancer invasion

Background: Hyaluronan (HA) mediates communication between cancer cells and the environment via interactions with the cell surface receptor CD44. We have previously shown that CD44-HA interaction in BC cells promotes adhesion to bone marrow endothelial cells. This suggests that HA/CD44 signalling may be correlated with breast cancer (BC) metastasis. The long-term objective of this study is to increase our understanding of the mechanisms by which CD44-HA interaction promotes BC metastasis, and further identify and validate CD44-downstream transcriptional targets for anti-metastatic therapy. Materials & Methods: Pursuant to this goal, we have developed a tetracycline (tet)-regulated expression of CD44 gene in the BC cell line MCF-7 (B5 clone) and identified TGF-β2 (Transforming Growth Factor beta-2; 3 fold induction) as a potential CD44s-downstream transcriptional target by microarray analysis. To further validate this finding, the same RNA samples, used for microarray analysis and their corresponding protein lysates collected from the BC cell line MCF-7-B5, were examined for CD44 expression in the presence of HA for 18, 24, and 48 hr post-tet withdrawal. Expression of TGF-β2 was examined using RT-PCR and western-Blot analyses. Results: Our results showed that TGF-β2 mRNA levels were significantly elevated following the removal of tet at 18, 24, and 48 h post-HA stimulation compared to the parental cells. Furthermore, the TGF-β2 precursor protein increased in a time-dependent pattern upon HA-stimulation and in the absence of tet. More interestingly, inhibition of CD44 gene by RNAi method decreased TGF-β2 expression upon HA-stimulation and in the absence of tet. Conclusion: Our data strongly support the hypothesis that TGF-β2 is a potential target of HA/CD44- downstream-signaling mediating BC cell invasion. Ongoing investigation aims to elucidate the signal transduction pathways coupling CD44 to the regulation of TGF-β2 expression, and further validate CD44/TGF-β2 axes in breast tumor invasion and metastasis. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-16.