Anupama Chandramouli

Loss of the Eukaryotic Initiation Factor 3f in Pancreatic Cancer

Aberrant regulation of the translation initiation is known to contribute to tumorigenesis. eIF3 plays an important role in translation initiation. eIF3f is the p47 subunit of the eIF3 complex whose function in cancer is not clear. Initial studies from our group indicated that eIF3f expression is decreased in pancreatic cancer. Overexpression of eIF3f induces apoptosis in pancreatic cancer cells. The eIF3f gene is located at chromosome band region 11p15.4. Loss of 11p15.4 is a common event in many tumors including pancreatic cancer. In order to investigate the molecular mechanism of the decreased expression of eIF3f in pancreatic cancer, we performed loss of heterozygosity (LOH) analysis in 32 pancreatic cancer specimens using three microsatellite markers encompassing the eIF3f gene. We showed that the prevalence of LOH ranged from 71% to 93%. We also performed eIF3f gene copy number analysis using quantitative real time PCR to further confirm the specific allelic loss of eIF3f gene in pancreatic cancer. We demonstrated a statistically significant decrease of eIF3f gene copy number in pancreatic tumors compared with normal tissues with a tumor/normal ratio of 0.24. Furthermore, RNA in situ hybridization and tissue microarray immunohistochemistry analysis demonstrated that eIF3f expression is significantly decreased in human pancreatic adenocarcinoma tissues compared to normal pancreatic tissues. These data provides new insight into the understanding of the molecular pathogenesis of eIF3f during pancreatic tumorigenesis.

Loss of the eukaryotic initiation factor 3f in melanoma

Aberrant regulation of the translation initiation is known to contribute to tumorigenesis. eIF3 plays an important role in translation initiation. eIF3f is the p47 subunit of the eIF3 complex whose function in cancer is not clear. Initial studies from our group indicated that eIF3f expression is decreased in melanoma. Overexpression of eIF3f inhibits translation and induces apoptosis in melanoma cells. The eIF3f gene is located at chromosome region 11p15.4. Loss of 11p15.4 is a common event in many tumors including melanoma. In order to investigate the molecular mechanism of the decreased expression of eIF3f in melanoma, we performed loss of heterozygosity (LOH) analysis in 24 melanoma specimens using three microsatellite markers encompassing the eIF3f gene. We showed that the prevalence of LOH ranged from 75% to 92% in melanoma. We also performed eIF3f gene copy number analysis using quantitative real‐time PCR to further confirm the specific allelic loss of the eIF3f gene in melanoma. We demonstrated a statistically significant decrease of the eIF3f gene copy number in melanoma compared with normal tissues with a tumor/normal ratio of 0.52. To further elucidate the somatic genetic alterations, we carried out mutation analysis covering the entire coding region and 5′UTR of the eIF3f gene in melanoma tissues and cell lines. Despite some polymorphisms, we did not find any mutations. Furthermore, immunohistochemistry analysis demonstrated that eIF3f protein expression is decreased in melanoma compared to benign nevi. These data provide new insight into the understanding of the molecular pathogenesis of eIF3f during melanoma tumorigenesis.

Disease modification of breast cancer-induced bone remodeling by cannabinoid 2 receptor agonists.

Most commonly originating from breast malignancies, metastatic bone cancer causes bone destruction and severe pain. Although novel chemotherapeutic agents have increased life expectancy, patients are experiencing higher incidences of fracture, pain, and drug‐induced side effects; furthermore, recent findings suggest that patients are severely undertreated for their cancer pain. Strong analgesics, namely opiates, are first‐line therapy in alleviating cancer‐related pain despite the severe side effects, including enhanced bone destruction with sustained administration. Bone resorption is primarily treated with bisphosphonates, which are associated with highly undesirable side effects, including nephrotoxicity and osteonecrosis of the jaw. In contrast, cannabinoid receptor 2 (CB2) receptor‐specific agonists have been shown to reduce bone loss and stimulate bone formation in a model of osteoporosis. CB2 agonists produce analgesia in both inflammatory and neuropathic pain models. Notably, mixed CB1/CB2 agonists also demonstrate a reduction in ErbB2‐driven breast cancer progression. Here we demonstrate for the first time that CB2 agonists reduce breast cancer–induced bone pain, bone loss, and breast cancer proliferation via cytokine/chemokine suppression. Studies used the spontaneously‐occurring murine mammary cell line (66.1) implanted into the femur intramedullary space; measurements of spontaneous pain, bone loss, and cancer proliferation were made. The systemic administration of a CB2 agonist, JWH015, for 7 days significantly attenuated bone remodeling, assuaged spontaneous pain, and decreased primary tumor burden. CB2‐mediated effects in vivo were reversed by concurrent treatment with a CB2 antagonist/inverse agonist but not with a CB1 antagonist/inverse agonist. In vitro, JWH015 reduced cancer cell proliferation and inflammatory mediators that have been shown to promote pain, bone loss, and proliferation. Taken together, these results suggest CB2 agonists as a novel treatment for breast cancer–induced bone pain, in which disease modifications include a reduction in bone loss, suppression of cancer growth, attenuation of severe bone pain, and increased survival without the major side effects of current therapeutic options.

The induction of S100p expression by the Prostaglandin E2 (PGE2)/EP4 receptor signaling pathway in colon cancer cells

Background: Prostaglandin E2 (PGE2) levels are frequently elevated in colorectal carcinomas. PGE2 is perceived via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is most relevant. PGE2/EP4-receptor interaction activates CREB via the ERK/MEK pathway. However, the downstream target genes activated by this pathway remained to be investigated. Methodology/Prinicipal Findings: Here, we have identified S100P (an EF-hand calcium binding protein) as a novel downstream target. We show by realtime RT-PCR that S100P mRNA levels are elevated in 14/17 (82%) colon tumor tissues as compared to paired adjacent normal colonic tissues. S100P expression is stimulated in the presence of PGE2 in a time dependent manner at mRNA and protein levels in colon, breast and pancreatic cancer cells. Pharmacological and RNAi-mediated inhibition of the EP4 receptor attenuates PGE2-dependent S100P mRNA induction. RNAi-mediated knockdown of CREB inhibits endogenous S100P expression. Furthermore, using luciferase reporter analysis and EMSA we show that mutation and/or deletion of the CRE sequence within the S100P promoter abolished PGE2-mediated transcriptional induction. Finally, we demonstrate that RNAi-mediated knockdown of S100P compromised invadopodia formation, colony growth and motility of colon cancer cells. Interestingly, endogenous knock down of S100P decreases ERK expression levels, suggesting a role for ERK in regulating S100P mediated cell growth and motility. Conclusions/Significance: Together, our findings show for the first time that S100P expression is regulated by PGE2/EP4-receptor signaling and may participate in a feedback signaling that perpetuates tumor cell growth and migration. Therefore, our data suggest that dysregulated S100P expression resulting from aberrant PGE2/EP4 receptor signaling may have important consequences relevant to colon cancer pathogenesis.

Inhibition of p38-MAPK signaling pathway attenuates breast cancer induced bone pain and disease progression in a murine model of cancer-induced bone pain

BackgroundMechanisms driving cancer-induced bone pain are poorly understood. A central factor implicated to be a key player in the process of tumorigenesis, osteoclastogenesis and nociception is p38 MAPK. We determined the role of p38 MAPK in a mouse model of breast cancer induced bone pain in which mixed osteolytic and osteoblastic remodeling occurs.ResultsIn cancer-treated mice, acute as well as chronic inhibition of p38 MAPK with SB203580 blocked flinching and guarding behaviors in a dose-dependent manner whereas no effect on thresholds to tactile stimuli was observed. Radiographic analyses of bones demonstrated that chronic inhibition of p38 MAPK reduced bone loss and incidence of spontaneous fracture in cancer-treated mice. Histological analysis of bones collected from mice treated with the p38 MAPK inhibitor showed complete absence of osteoblastic growth in the intramedullary space as well as significantly reduced tumor burden.ConclusionsBlockade of non-evoked pain behaviors but not hypersensitivity suggests differences in the underlying mechanisms of specific components of the pain syndrome and a possibility to individualize aspects of pain management. While it is not known whether the role of p38 MAPK signaling can be expanded to other cancers, the data suggest a need for understanding molecular mechanisms and cellular events that initiate and maintain cancer-induced bone pain for effective management for both ongoing pain as well as breakthrough pain.

Modulation of breast cancer cell viability by a cannabinoid receptor 2 agonist, JWH-015, is calcium dependent.

Introduction Cannabinoid compounds, both nonspecific as well as agonists selective for either cannabinoid receptor 1 (CB1) or cannabinoid receptor 2 (CB2), have been shown to modulate the tumor microenvironment by inducing apoptosis in tumor cells in several model systems. The mechanism of this modulation remains only partially delineated, and activity induced via the CB1 and CB2 receptors may be distinct despite significant sequence homology and structural similarity of ligands. Methods The CB2-selective agonist JWH-015 was used to investigate mechanisms downstream of CB2 activation in mouse and human breast cancer cell lines in vitro and in a murine mammary tumor model. Results JWH-015 treatment significantly reduced primary tumor burden and metastasis of luciferase-tagged murine mammary carcinoma 4T1 cells in immunocompetent mice in vivo. Furthermore, JWH-015 reduced the viability of murine 4T1 and human MCF7 mammary carcinoma cells in vitro by inducing apoptosis. JWH-015-mediated reduction of breast cancer cell viability was not dependent on Gαi signaling in vitro or modified by classical pharmacological blockade of CB1, GPR55, TRPV1, or TRPA1 receptors. JWH-015 effects were calcium dependent and induced changes in MAPK/ERK signaling. Conclusion The results of this work characterize the actions of a CB2-selective agonist on breast cancer cells in a syngeneic murine model representing how a clinical presentation of cancer progression and metastasis may be significantly modulated by a G-protein-coupled receptor.

Abstract 2300: MicroRNA-101 (miR-101) posttranscriptionally regulates the expression of EP4 receptor in colon cancers

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The predominant product of cyclooxygenase (COX-2) in the colon, prostaglandin (PG) E2 promotes carcinogenesis. Expression of the PGE2 receptor EP4 is upregulated during colorectal carcinogenesis. However the mechanism leading to deregulation of the EP4 receptor is not known. The present study was conducted to investigate the regulation of EP4 receptor by miRNAs. A bioinformatics search revealed a conserved target site for miR-101 within the EP4 receptor-3′ UTR. In both colorectal cancer cell lines and human specimens, we observed an inverse correlation between miR-101 and EP4 receptor protein. Transfection of LS174T cells with miR-101 significantly suppressed a luciferase reporter containing the EP4 receptor-3′-UTR. In contrast, a mutant EP4 receptor-3′-UTR was unaffected. Ectopic expression miR-101 markedly reduced cell proliferation and motility. Co-transfection of EP4 receptor could rescue colon cancer cells from the tumor suppressive effects of miR-101. Moreover, pharmacologic inhibition of EP4 receptor signaling or silencing of EP4 receptor phenocopied the effect of miR-101. This is the first study to show that the EP4 receptor is negatively regulated by miR-101. 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 2300. doi:1538-7445.AM2012-2300

Abstract 2082: The microRNA-101 regulates the expression of EP4 receptor in colon cancers

Colorectal cancer (CRC) is one of the most devastating cancers in the United States and is characterized by the accumulation of prostaglandin PGE 2 . PGE 2 exerts its biological function via four transmembrane G protein coupled receptors (EP1-4), among which the prostanoid EP4 receptor is the most relevant. However, its expression in human CRC specimens has not been fully understood. Therefore, we wanted to clarify the expression of EP4 receptor in human tissue specimens. We collected a total of 40 CRC tissue specimens representing different stages of disease and performed immunohistochemistry (IHC) analysis. The specimens were graded for intensity and extent of staining of the EP4 receptor. Here we show that cancer tissues (n = 15) expressed significantly higher EP4 receptor staining as compared to normal colonic (n = 16) and adenoma (n = 9) tissues. We also performed western blot analysis on paired cancer and normal tissue specimens from this cohort and confirmed that the EP4 protein levels were elevated in cancer as compared to normal tissues whereas there was no difference in the expression of EP2 receptor. By contrast, the mRNA expression of the EP4 receptor gene (PTGER4) was down-regulated in these tumors as compared to paired normal tissue. We postulated that the increased EP4 receptor protein levels observed in tumors occurs by post transcriptional mechanisms. Therefore, to address this issue, we performed in silico analysis of the 3′UTR of PTGER4 gene for microRNA binding sites. We identified a consensus microRNA-101 (miR-101) seed sequence within the 3′UTR, proximal to the coding region, of the PTGER4 gene. Interestingly we observed a correlation between loss of miR-101 levels and increase in EP4 receptor protein levels in human CRC tissue specimens as compared to paired normal colonic tissue, by RT-PCR analysis. In order to directly assess this correlation, we ectopically expressed mir-101 plasmid in colon cancer cells and demonstrated that it could suppress endogenous levels of EP4 receptor protein. Together, these findings show that EP4 receptor is significantly elevated in human CRC and suggest, for the first time, that miR-101 may negatively regulate the expression of EP4 receptor by post-transcriptional mechanisms. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2082.

Abstract P1-11-23: Cannabinoid Receptor 2 Compounds in the Attenuation of Breast Cancer Cell Proliferation: Mechanisms of Action

Cannabinoids have been well established as mediators of tumor cell proliferation in several cancer models. The activity of cannabinoid receptor 1 (CB1) agonists as well as cannabinoid receptor 2 (CB2) agonists have been extensively studied over the last decade, though their mechanisms of action have yet to be defined in the context of attenuating tumor proliferation. CB1 is abundant in the central nervous system with a low level of expression in the periphery, while CB2 exists primarily on cells of the immune system. Due to the lack of neuronal expression of CB2 receptors, compounds acting at CB2 receptors do not produce the psychotropic effects associated with CB1 receptor compounds. Although CB1 and CB2 compounds have yielded similar antiproliferative results in some tumor cells in vitro, CB2 receptors are markedly upregulated in many tumor cell lines for unknown reasons. Therefore, we focused on compounds selective for the CB2 receptor, including the CB2 selective agonists JWH-015 and AM1241. We show here that CB2 agonists and antagonists alike attenuate the proliferation of mouse and human breast cancer cells in a concentration dependent manner. CB2 agonist induced breast cancer anti-proliferation is not blocked by pretreatment with pertussis toxin, the CB1 cannabinoid receptor antagonist SR141716, or the transient receptor potential cation channel subfamily V member 1 (TRPV1) antagonist capsazepine. The CB1 and CB2 receptors are classified as G-protein coupled receptors (GPCRs) that are generally linked to Gai or Gaq. Agonists and antagonists of G protein coupled receptors are typically defined based on their Ga mediated effect on intracellular cAMP levels. This method of classification is not entirely effective: it ignores the possibility of differential effects on alternative kinases and the bg subunit activity. The anti-proliferative activity of both JWH-015 and SR144528 may be explained by alternative receptor coupling pathways, changes in constitutive activity, or activity at a separate receptor. Here, we show a dose and time dependent decrease in phosphorylated ERK in cells treated with either JWH-015 (CB2 agonist) or SR144528 (CB2 antagonist). Together, these data along with the absence of a pertussis toxin effect suggest that the CB2 compounds are acting in a non-Gai coupled manner, and are attenuating a pro-survival pathway. Further studies are necessary to identify the binding partner responsible for the antiproliferative effects of CB2 compounds. We advance the idea that CB2 receptors on breast cancer cells constitutively activate the MAP/ERK pro-survival pathway and that by preventing constituitive activity of the CB2 receptor, CB2 compounds downregulate phosphorylation of the MAPK/ERK pathway to promote apoptosis of breast cancer cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-11-23.