Sanaa M. Nabha

Role of MAP kinase in tumor progression and invasion.

Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is a frequent event in tumorigenesis. MAPKs have been implicated in cell migration, proteinase-induction, regulation of apoptosis, and angiogenesis, events that are essential for successful completion of metastasis. In this review, we discuss the potential role that MAPKs play in metastasis by regulating cell migration, proteinase-induction and apoptosis.

Upregulation of PKC-δ contributes to antiestrogen resistance in mammary tumor cells

Acquired resistance to tamoxifen (Tam) in breast cancer patients is a serious therapeutic problem. We have previously reported that protein kinase C-delta (PKC-δ) plays a major role in estrogen (E2)-mediated cell proliferation. To determine if PKC-δ is one of the major alternate signaling pathways that supports cell growth in the presence of Tam, we determined the levels of PKC isoforms in four different models of antiestrogen-resistant cells. Three out of four antiestrogen resistance cell lines (Tam/MCF-7, ICI/MCF-7 and HER-2/MCF-7) expressed significantly high levels of both total and activated PKC-δ levels compared to sensitive cells. Estrogen receptor (ER) alpha content and function are maintained in all the antiestrogen-resistant cell lines. Overexpressing active PKC-δ in Tam-sensitive MCF-7 cells (PKC-δ/MCF-7) led to Tam resistance both in vitro and in vivo. Inhibition of PKC-δ by rottlerin (a relatively specific inhibitor of PKC-δ) or siRNA significantly inhibited estrogen- and Tam-induced growth in antiestrogen-resistant cells. PKC-δ levels are significantly higher in Tam-resistant tumors compared to Tam-sensitive tumors in xenograft model (P<0.05). Taken together, these data suggest that PKC-δ plays a major role in antiestrogen resistance in breast tumor cells and thus provides a new target for treatment.

Inhibition of human prostate cancer growth, osteolysis and angiogenesis in a bone metastasis model by a novel mechanism-based selective gelatinase inhibitor

Metastasis to the bone is a major clinical complication in patients with prostate cancer (PC). However, therapeutic options for treatment of PC bone metastasis are limited. Gelatinases are members of the matrix metalloproteinase (MMP) family and have been shown to play a key role in PC metastasis. Herein, we investigated the effect of SB‐3CT, a covalent mechanism‐based MMP inhibitor with high selectivity for gelatinases, in an experimental model of PC bone metastases. Intraperitoneal (i.p.) treatment with SB‐3CT (50 mg/kg) inhibited intraosseous growth of human PC3 cells within the marrow of human fetal femur fragments previously implanted in SCID mice, as demonstrated by histomorphometry and Ki‐67 immunohistochemistry. The anti‐osteolytic effect of SB‐3CT was confirmed by radiographic images. Treatment with SB‐3CT also reduced intratumoral vascular density and bone degradation in the PC3 bone tumors. A direct inhibition of bone marrow endothelial cell invasion and tubule formation in Matrigel by SB‐3CT in vitro was also demonstrated. The use of the highly selective gelatinase inhibitors holds the promise of effective intervention of metastases of PC to the bone.

Bone marrow stromal cells enhance prostate cancer cell invasion through type I collagen in an MMP‐12 dependent manner

At the cellular level, the process of bone metastasis involves many steps. Circulating cancer cells enter the marrow, proliferate, induce neovascularization, and ultimately expand into a clinically detectable, often symptomatic, metastatic deposit. Although the initial establishment and later expansion of the metastatic deposit in bone require tumor cells to possess invasive capability, the exact proteases responsible for this phenotype are not well known. The objective of our study was to take an unbiased approach to determine which proteases were expressed and functional during the initial interactions between prostate cancer cells and bone marrow stromal (BMS) cells. We found that the combination of human prostate cancer PC3 and BMS cells stimulates the invasive ability of cancer cells through type I collagen. The use of inhibitors for each of the major protease families indicated that 1 or more MMPs was/were responsible for the BMS‐induced invasion. Gene profiling and semiquantitative RT‐PCR analysis revealed an increased expression of several MMP genes because of PC3/BMS cell interaction. However, only MMP‐12 showed an increase in protein expression. Downregulation of MMP‐12 expression in PC3 cells by siRNA inhibited the enhanced invasion induced by PC3/BMS cell interaction. In vivo, MMP‐12 was found to be primarily expressed by prostate cancer cells growing in bone. Our data suggest that BMS cells induce MMP‐12 expression in prostate cancer cells, which results in invasive cells capable of degradation of type I collagen.

Effects of combretastatin A-4 prodrug against a panel of malignant human B-lymphoid cell lines.

Combretastatin A-4 (CA-4) is one of a family of compounds isolated from the South African willow tree Combretum caffrum. CA-4 was found to be active against murine melanoma and a variety of other human solid tumors. For the first time, we report the effect of CA-4 against a panel of malignant human B-lymphoid cell lines [early pre-B acute lymphoblastic leukemia (Reh), diffuse large cell lymphoma (WSU-DLCL2), chronic lymphocytic leukemia (WSU-CLL) and Waldenstroms macroglobulinemia (WSU-WM)]. Our results indicate, using the prodrug form of CA-4, a concentration-dependent growth inhibition in all tested cell lines, although WSU-DLCL2 was more sensitive. Exposure to 4 nM CA-4 for 96 h induced 77% growth inhibition in Reh, 86% in WSU-CLL and 92% in WSU-WM. When used against the WSU-DLCL2 cell line, this same concentration of CA-4 was completely toxic. Morphological examination showed CA-4 induced the formation of giant, multinucleated cells, a phenomenon commonly found in mitotic catastrophe. Only minimal numbers of cells showing characteristics of apoptosis were detected. In WSU-DLCL2 cells, CA-4 (3 nM) induced the highest apoptosis (5%) after 48 h, while the percentage of dead cells was approximately 47%. Exposure of Reh, WSU-CLL, WSU-WM and WSU-DLCL2 cells for 24 h to 5 nM CA-4 induced 19, 28, 57 and 75% G2/M arrest, as determined by flow cytometry, respectively. Based on these preliminary studies, we believe that mitotic catastrophe is the predominant mechanism by which CA-4 induces cell death rather than apoptosis. Further studies to elucidate the mechanisms of CA-4 activity in vitro and in vivo are currently under investigation in our laboratory.

Host matrix metalloproteinase-9 contributes to tumor vascularization without affecting tumor growth in a model of prostate cancer bone metastasis

Matrix metalloproteinases (MMPs) have been associated with initiation, progression and vascularization of a number of tumors. However, clinical trials using MMP inhibitors failed to meet expectations. Previously, we demonstrated the potential importance of MMP-9 activity in experimental prostate cancer bone tumor tissue. However, the particular roles of host- and tumor-derived MMP-9 remains to be defined. Herein, we examined the role of host MMP-9 in subcutaneous and intraosseous growth of the human androgen independent prostate cancer cell line PC3 in MMP-9 deficient mice. In the subcutaneous model, the tumor incidence in the control (RAG-1ko/ko) and experimental (RAG-1ko/ko /MMP-9ko/ko) group was 100%, with similar tumor growth kinetics and microvascular densities. In the intraosseous tumor model, the tumor incidence was higher in RAG-1ko/ko /MMP-9ko/ko mice than in RAG-1ko/ko mice (67% and 39%, respectively), though no statistical differences were found. The intraosseous tumor areas were similar in both groups, and the number of tumor-associated osteoclasts did not differ significantly. However, the microvascular density of intraosseous tumors was higher in RAG-1ko/ko than in RAG-1ko/ko/MMP-9ko/ko mice, though no changes in tumor growth could be detected. In an in vitro assay, we found that bone marrow (BM) cells increased the invasiveness of PC3 cells, and that this enhancement was independent of MMP-9 expression by marrow cells. Our results with the RAG-1 model suggest that host-derived MMP-9 is neither necessary nor sufficient for subcutaneous or intraosseous PC3 tumor growth, osteoclastic response, or in vitro invasiveness of tumor cells.

Evaluation of combretastatin A-4 prodrug in a non-Hodgkin's lymphoma xenograft model: preclinical efficacy.

Combretastatin A-4 prodrug (CA4P) is a new antitubulin agent currently in phase I/II clinical trials against solid tumors. We have previously reported on the in vitro activity of CA4P against a panel of malignant human B-lymphoid cell lines. In this study, we investigated the antitumor and the antiangiogenic activity of CA4P in our diffuse large cell lymphoma WSU-DLCL2-SCID mouse model. WSU-DLCL2 cells (107) were injected s.c. into 5-week-old female ICR-SCID mice. Tumor-bearing mice were treated at the CA4P maximum tolerated dose (MTD) of 800 mg/kg in different dose/schedules. CA4P showed significant antitumor activity against this lymphoma model. Best results were seen when MTD was given in two and four divided doses (400 and 200 mg/kg, respectively). CA4P given in four divided doses (4×200 mg/kg) showed a log10 kill of 1.01, T/C of 11.7% and T-C of 12 days. Immunohistochemical staining using anti-CD31 antibody after 6, 24, 48 and 120 h treatment revealed a significant decrease in the number of tumor blood vessels after 24 h (about 80%). Only the periphery of treated tumors revealed the presence of blood vessels. Morphological examination of the tumors after tetrachrome staining showed a necrotic center in tumors of CA4P-treated animals. New blood vessel formation was noted to emerge in tumor tissues as early as 48 h following a single dose of CA4P. The G2/M arrest observed in vitro was not detected in vivo indicating predominance of the antiangiogenic effects with regard to antitumor efficacy in vivo. We conclude that CA4P has antiangiogenic activity in this lymphoma model and the use of this agent should be explored clinically in the treatment of non-Hodgkins lymphoma.

Rituximab, Cyclophosphamide, Dexamethasone (RCD) regimen induces cure in WSU-WM xenograft model and a partial remission in previously treated Waldenstrom's macroglobulinemia patient.

Waldenstroms macroglobulinemia (WM) is an uncommon lymphoproliferative disease which remains incurable with current treatment protocols. We have previously established a permanent WM cell line, WSU-WM, which grows as a xenograft in severe combined immune deficient (SCID) mice. In this study, we investigated the antitumor effects of Rituximab (RTX), Cyclophosphamide (CTX), Dexamethasone (DEX) [RCD]-Regimen in vivo WSU-WM SCID xenograft and in a patient with WM. For the pre-clinical efficacy study, WSU-WM-bearing SCID mice were randomly assigned to receive RTX (150 mg/kg/inj, i.v., QDX5), CTX (90 mg/kg/inj, s.c. QDX5) as single agents or diluent. The combination group received RTX at 150 mg/kg/inj, QDX5; CTX at 150 mg/kg/inj, QODX3 and DEX at 1.0 mg/kg/inj, i.v., QDX5. Tumor growth inhibition (T / C) , tumor growth delay (T – C) , and log 10 kill (net) for RTX and CTX were 24.5%, 37 days, 5.52 and 88%, 0.0 days, 0.0 log 10 kill, respectively. No cures were observed with either agent; however, all mice (6/6, with bilateral tumors) were cured when treated with RCD-regimen. A 57-year-old patient with relapsed WM was treated with the RCD-regimen and showed an excellent partial remission for seven months. The patient tolerated the treatment very well, the hemoglobin improved dramatically, platelets remained stable, the IgM level normalized and there was only minimal involvement of bone marrow. Based on these results, the RCD regimen is effective against WM and its activity should be further evaluated in clinical trials.

Abstract 1439: De novo expression of c-kit in prostate cancer cells interacting with the bone microenvironment

Specific cellular and molecular interactions between prostate cancer (PC) cells and the bone microenvironment lead to expansion of the metastatic deposit, disease progression, and ultimately significant morbidity and mortality. We recently reported an association between expression of the tyrosine kinase receptor c-kit and clinical PC progression, with the highest expression seen in clinical metastatic bone specimens. Moreover, we found that intraosseous tumors formed by otherwise c-kit negative PC3 cells injected into human bone xenografts in SCID mice (SCID-hu model) expressed c-kit. To determine the relevance of these findings, we examined additional human PC cell lines, namely LNCaP, C42b, and DU145, for the expression of c-kit in vitro and in vivo. While none of the cultured PC cells expressed c-kit at the message or protein level, immunostaining for c-kit in intraosseous tumors produced by these cells in the SCID-hu model revealed a distinctive and specific reactivity in the cancer cell compartment. The induction of c-kit expression in PC cells growing within the bone microenvironment was emulated by co-culture of PC3 cells and bone-derived cells embedded in Matrigel, as demonstrated by RT-PCR analysis. In an attempt to mimic the de novo c-kit expression observed in the SCID-hu model and to identify potential signaling pathways triggered by c-kit activation by its ligand stem cell factor (SCF), normally expressed by the bone microenvironment, we transiently transfected PC3 cells with wild-type c-kit. Activation of c-kit was confirmed by immunoblotting for phosphorylated c-kit. Using the whole human genome Agilent microarray, we found that 220 genes were differentially expressed when SCF-treated and non-treated c-kit-transfected PC3 cells were compared, resulting in discrete functional sub-networks that were coordinately up or down-regulated. Most notably, pathway and genetic network analysis revealed that c-kit activation resulted in an up-regulated fibroblast growth factor-2-mediated sub-network. Taken together, these results suggest that c-kit induction in PC cells in the bone microenvironment modulates cell-cell interaction pathways and growth factor signaling systems that ultimately lead to expansion of bone metastasis. 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 1439.