Guillermo Peluffo

Role of COX-2 in epithelial–stromal cell interactions and progression of ductal carcinoma in situ of the breast

Epithelial–stromal cell interactions have an important role in breast tumor progression, but the molecular mechanisms underlying these effects are just beginning to be understood. We previously described that fibroblasts promote, whereas normal myoepithelial cells inhibit, the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinomas by using a xenograft model of human DCIS. Here, we report that the tumor growth and progression-promoting effects of fibroblasts are at least in part due to increased COX-2 expression in tumor epithelial cells provoked by their interaction with fibroblasts. Up-regulation of COX-2 in DCIS xenografts resulted in increased VEGF and MMP14 expression, which may contribute to the larger weight and invasive histology of COX-2-expressing tumors. Administration of celecoxib, a selective COX-2 inhibitor, to tumor-bearing mice decreased xenograft tumor weight and inhibited progression to invasion. Coculture of fibroblasts with DCIS epithelial cells enhanced their motility and invasion, and this change was associated with increased MMP14 expression and MMP9 protease activity. We identified the NF-κB pathway as one of the mediators of stromal fibroblast-derived signals regulating COX-2 expression in tumor epithelial cells. Inhibition of NF-κB and COX-2 activity and down-regulation of MMP9 expression attenuated the invasion-promoting effects of fibroblasts. These findings support a role for COX-2 in promoting the progression of DCIS to invasive breast carcinomas, and suggest that therapeutic targeting of the NF-κB and prostaglandin signaling pathways might be used for the treatment and prevention of breast cancer.

JARID1B Is a Luminal Lineage-Driving Oncogene in Breast Cancer

Recurrent mutations in histone-modifying enzymes imply key roles in tumorigenesis, yet their functional relevance is largely unknown. Here, we show that JARID1B, encoding a histone H3 lysine 4 (H3K4) demethylase, is frequently amplified and overexpressed in luminal breast tumors and a somatic mutation in a basal-like breast cancer results in the gain of unique chromatin binding and luminal expression and splicing patterns. Downregulation of JARID1B in luminal cells induces basal genes expression and growth arrest, which is rescued by TGFβ pathway inhibitors. Integrated JARID1B chromatin binding, H3K4 methylation, and expression profiles suggest a key function for JARID1B in luminal cell-specific expression programs. High luminal JARID1B activity is associated with poor outcome in patients with hormone receptor-positive breast tumors.

Reduction of tumor progression and paraneoplastic syndrome development in murine lung adenocarcinoma by nonsteroidal antiinflammatory drugs

Mice bearing LP07 lung adenocarcinoma show some characteristics that are similar to those present in patients with NSCLC. LP07 tumor‐bearing mice develop the paraneoplastic syndromes of cachexia, leukocytosis and hypercalcemia. These symptoms may be partly due to a systemic inflammatory response. Our aim was to determine if treatment with NSAIDs would lower tumor and metastasis growth and their accompanying syndromes. The nonselective COX inhibitor indomethacin and the selective COX‐2 inhibitor celecoxib reduced tumor growth and metastasis outcome in s.c. LP07 tumor‐bearing mice. Both drugs also inhibited the development of leukocytosis and the weight loss associated with LP07 progression. Serum levels of the inflammatory cytokines IL‐1β and IL‐6, mediators of cachexia, were modulated by NSAIDs. Inhibition of in vitro migration and invasion and reduction in angiogenesis were attained when cells were treated with either indomethacin or celecoxib. MMP‐9 activity was also reduced in conditioned media from LP07 cells treated with celecoxib. These data suggest that several processes implicated in tumor progression can be modulated with NSAID treatment. Improvement in performance status through modulation of cachexia may offer a possibility for combining anti‐inflammatory treatments with more aggressive therapies.

Inhibition of Tumor Progression and Paraneoplastic Syndrome Development in a Murine Lung Adenocarcinoma by Medroxyprogesterone Acetate and Indomethacin

Mice bearing LP07 lung adenocarcinoma present some characteristics similar to those shown in patients with several malignant diseases. LP07 tumor bearers develop paraneoplastic syndromes such as cachexia, leukocytosis, and hypercalcemia, partly due to a systemic inflammatory response. We analyzed some of the mechanisms involved in the effectiveness of the association of the appetite-stimulant medroxiprogesterone acetate (MPA) and the nonselective cyclooxigenase (COX) inhibitor indomethacin (INDO) in LP07 tumor bearing mice. INDO and INDO plus MPA treatments significantly inhibited tumor growth, which was not inhibited by MPA. The number of lung metastatic nodules was decreased with all treatments, being most effective INDO alone and INDO plus MPA. A significant decrease of plasmatic levels of the matrix metalloproteinases MMP-9 and MMP-2 correlated with these results. Paraneoplastic syndromes, leukocytosis, and cachexia were abolished by all treatments. We determined effects of the treatments on circulating cytokines shown to regulate cachexia and inflammation. Both treatments alone, and INDO plus MPA, reduced circulating IL-6 throughout tumor evolution. A pronounced increase in serum IL-1β levels was detected in untreated tumor bearers. These levels decreased and were closer to normal serum values when LP07 mice were treated with INDO plus MPA. The combination of a nonsteroidal antiinflammatory drug as INDO and MPA showed to be effective in inhibiting tumor and metastatic growth and diminishing paraneoplastic symptoms and SIR. A variety of specific molecules are implicated as playing a role in cancer-induced cachexia and hematological alterations.

Abstract 2231: The over-activation of Akt1 oncogene heightens the Glypican-3 (GPC3) effects on metastatic progression of murine mammary tumor cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL It is known that Akt kinases 1, 2 and 3 have critical roles in regulating survival, metabolism and other cellular activities. However, their functions on migratory potential and metastasis development are less clear. GPC3 is a proteoglycan downregulated in breast tumors. We have previously shown that GPC3 reexpression inhibits metastatic capacity in vivo of the LM3 murine mammary adenocarcinoma cells. Moreover, this phenomenon was associated with a partial reversion of the epithelial-to-mesenchymal transition as shown in vitro. On the other hand, we have also determined that GPC3 modulates Akt signaling pathway in LM3 cells. The aim of this work was to investigate the role of Akt1 on the invasive/metastatic behavior of GPC3 reexpressing cells. First, we have confirmed that LM3-GPC3 cells present higher Akt1 levels than LM3-vector ones by western blot. To elucidate the function of this isoform, LM3-GPC3 clones were infected with viruses carrying a constitutively active variant of Akt1 (CA Akt1). Although there were no changes in the actin cytoskeleton organization, we could determine by a wound healing assay that LM3-GPC3-CA Akt1 cells were less migrant than their controls (Wound coverage: LM3-GPC3 control=50% vs. LM3-GPC3-CA Akt1=30%, p<0.05). To address an in vivo effect, different clones were injected s.c. into syngeneic female BALB/c mice. We found that these tumors did not differ in their growth rate, reaching a similar size at 30 days post inoculation (LM3-vector= 189±25, LM3-GPC3= 181±40, LM3-GPC3-CA Akt1= 200±51 mm3). No differences were found in either tumor incidence or in tumor latency. In order to study the experimental metastatic ability, all cell lines were i.v. inoculated. We observed that LM3-GPC3-CA Akt1 tumors developed fewer lung metastases than controls (Median [Range]: LM3-GPC3 control=9 [3-29] vs. LM3-GPC3 CA Akt1=0.5 [0-10], p<0.05). Moreover, the LM3-GPC3-CA Akt1 metastatic nodes were also of a smaller size (<0.5 mm). In sum, our results suggest that Akt1 expression and signaling could be mediating GPC3 inhibitory effect on the metastatic dissemination. The over-activation of this oncogene would exacerbate or heighten GPC3 effects, preventing the metastasis progression through the reduction of cell migration. 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 2231. doi:1538-7445.AM2012-2231

Abstract 2900: COX-2 modulates murine lung adenocarcinoma cell survival by activating Akt and NF-κB pathways and inhibiting p38MAPK

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Cyclooxygenase-2 (COX-2) expression is increased in a number of tumors along with elevated prostaglandin (PG) production. The treatment with COX-2 inhibitors was found to have antitumor properties in different types of cancers, although the dependency of COX-2 inhibition remains controversial. We aimed to study the role of COX-2 in the progression of a murine lung adenocarcinoma. COX-2-specific inhibitor celecoxib delayed tumor growth (p < 0.001 by day 30 after subcutaneous tumor inoculation) and the metastatic outcome of LP07 cells. The treatment with celecoxib reduced LP07 cell motility, invasiveness and the activity of secreted matrix metalloproteinase-2 (MMP-2). Celecoxib (25 μM) also increased the susceptibility of LP07 cells to serum deprivation (p < 0.001) and exogenously added PGE2 (10 μM) counteracted this effect. The reduced viability of tumor cells by specific COX-2 inhibition correlated with decreased levels (2.25 fold) of phosphorylated Akt in serum-stimulated LP07 cells and a reduced activity of NF-κB (p < 0.001), a transcription factor with survival/anti-apoptotic properties. We also found increased levels of phosphorylated p38 MAPK in serum-starved LP07 cells treated with celecoxib. The effect of COX-2 inhibition on cell viability was partially abolished by infecting LP07 cells with a retroviral vector coding for a constitutively active form of Akt or by transfection with a dominant negative p38. The activation of the Akt pathway partially reverted the inhibition of NF-κB activity. To further study the role of COX-2 in LP07 tumor biology, we inhibited COX-2 expression by infection with a retroviral vector with specific shRNAs. Clones with the lowest COX-2 expression were selected. Controls expressed a shRNA for GFP or carried an empty vector. The inhibition of COX-2 expression resulted in reduced tumor growth in mice (p < 0.001 in all cases compared to the shGFP control). Homogenates from these tumors showed a low level of phosphorylated Akt, in line with in vitro experiments where LP07 cells were less responsive to serum pulses in phosphorylating Akt if COX-2 expression was blocked. The inhibition of COX-2 expression reduced the viability of tumor cells after serum depletion (p < 0.01), and this effect was completely abolished by PGE2 and partially by the expression of a constitutively active form of Akt and PI3K, or a dominant negative p38. NF-κB activity was also reduced by the inhibition of COX-2 expression (p < 0.01). Taken together, we found that COX-2 is a key player for the malignant behavior of the LP07 lung tumor. COX-2 and PGE2 had an important role for LP07 cell survival by modulating the Akt and p38 MAPK and NF-κB pathways 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 2900. doi:10.1158/1538-7445.AM2011-2900