Kristen Anderson

A Novel Lung Metastasis Signature Links Wnt Signaling with Cancer Cell Self-Renewal and Epithelial-Mesenchymal Transition in Basal-like Breast Cancer

The establishment of metastasis depends on the ability of cancer cells to acquire a migratory phenotype combined with their capacity to recreate a secondary tumor in a distant tissue. In epithelial cancers, such as those of the breast, the epithelial-mesenchymal transition (EMT) is associated with basal-like breast cancers, generates cells with stem-like properties, and enables cancer cell dissemination and metastasis. However, the molecular mechanism(s) that connects stem cell-like characteristics with EMT has yet to be defined. Using an orthotopic model of human breast cancer metastasis to lung, we identified a poor prognosis gene signature, in which several components of the wnt signaling pathway were overexpressed in early lung metastases. The wnt genes identified in this signature were strongly associated with human basal-like breast cancers. We found that inhibiting wnt signaling through LRP6 reduced the capacity of cancer cells to self-renew and seed tumors in vivo. Furthermore, inhibition of wnt signaling resulted in the reexpression of breast epithelial differentiation markers and repression of EMT transcription factors SLUG and TWIST. Collectively, these results provide a molecular link between self-renewal, EMT, and metastasis in basal-like breast cancers.

Human bone marrow-derived MSCs can home to orthotopic breast cancer tumors and promote bone metastasis.

American women have a nearly 25% lifetime risk of developing breast cancer, with 20% to 40% of these patients developing life-threatening metastases. More than 70% of patients presenting with metastases have skeletal involvement, which signals progression to an incurable stage. Tumor-stroma cell interactions are only superficially understood, specifically regarding the ability of stromal cells to affect metastasis. In vivo models show that exogenously supplied human bone marrow-derived stem cells (hBMSC) migrate to breast cancer tumors, but no reports have shown endogenous hBMSC migration from the bone to primary tumors. Here, we present a model of in vivo hBMSC migration from a physiologic human bone environment to human breast tumors. Furthermore, hBMSCs alter tumor growth and bone metastasis frequency. These may home to certain breast tumors based on tumor-derived TGF-β1. Moreover, at the primary tumor level, interleukin 17B (IL-17B)/IL-17BR signaling may mediate interactions between hBMSCs and breast cancer cells.

Tissue-Engineered Bone Serves as a Target for Metastasis of Human Breast Cancer in a Mouse Model

The high frequency and mortality associated with breast cancer metastasis to bone has motivated efforts to elucidate tumor-stroma interactions in the bone microenvironment contributing to invasion and proliferation of metastatic cells. The development of engineered tissues has prompted the integration of engineered bone scaffolds into animal models as potential targets for metastatic spread. Silk scaffolds were coupled with bone morphogenetic protein-2 (BMP-2), seeded with bone marrow stromal cells (BMSC), and maintained in culture for 7 weeks, 4 weeks, and 1 day before s.c. implant in a mouse model of human breast cancer metastasis from the orthotopic site. Following injection of SUM1315 cells into mouse mammary fat pads, tumor burden of implanted tissues was observed only in 1-day scaffolds. Scaffold development and implantation was then reinitiated to identify the elements of the engineered bone that contribute to metastatic spread. Untreated scaffolds were compared with BMP-2-coupled, BMSC-seeded, or BMP-2/BMSC-combined treatment. Migration of SUM1315 cells was detected in four of four mice bearing scaffolds with BMP-2 treatment and with BMSC treatment, respectively, whereas only one of six mice of the BMP-2/BMSC combination showed evidence of metastatic spread. Histology confirmed active matrix modeling and stromal cell/fibroblast infiltration in scaffolds positive for the presence of metastasis. These results show the first successful integration of engineered tissues in a model system of human breast cancer metastasis. This novel platform now can be used in continued investigation of the bone environment and stem cell contributions to the process of breast cancer metastasis.

Differential Response of Human Ovarian Cancer Cells to Induction of Apoptosis by Vitamin E Succinate and Vitamin E Analogue, α-TEA

A vitamin E derivative, vitamin E succinate (VES; RRR-α-tocopheryl succinate), and a vitamin E analogue, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy acetic acid (α-TEA), induce human breast, prostate, colon, lung, cervical, and endometrial tumor cells in culture to undergo apoptosis but not normal human mammary epithelial cells, immortalized, nontumorigenic breast cells, or normal human prostate epithelial cells. Human ovarian and cervical cancer cell lines are exceptions, with α-TEA exhibiting greater proapoptotic effects. Although both VES and α-TEA can induce A2780 and subline A2780/cp70 ovarian cancer cells to undergo DNA synthesis arrest within 24 h of treatment, only α-TEA is an effective inducer of apoptosis. VES or α-TEA treatment of cp70 cells with 5, 10, or 20 μg/ml for 3 days induced 5, 6, and 19% versus 9, 36, and 71% apoptosis, respectively. Colony formation data provide additional evidence that cp70 cells are more sensitive to growth inhibition by α-TEA than VES. Differences in stability of the ester-linked succinate moiety of VES versus the ether-linked acetic acid moiety of α-TEA were demonstrated by high-performance liquid chromatography analyses that showed α-TEA to remain intact, whereas VES was hydrolyzed to the free phenol, RRR-α-tocopherol. Pretreatment of cp70 cells with bis-(p-nitrophenyl) phosphate, an esterase inhibitor, before VES treatment, resulted in increased levels of intact VES and apoptosis. Taken together, these data show α-TEA to be a potent and stable proapoptotic agent for human ovarian tumor cells and suggest that endogenous ovarian esterases can hydrolyze the succinate moiety of VES, yielding RRR-α-tocopherol, an ineffective apoptotic-inducing agent.

Comparison of Vitamin E Derivatives α-TEA and VES in Reduction of Mouse Mammary Tumor Burden and Metastasis

A novel nonhydrolyzable ether derivative of RRR-α-tocopherol, RRR-α-tocopherol ether acetic acid analog [2, 5, 7, 8-tetramethyl-2R-(4R, 8R, 12-trimethyltridecyl)chroman-6-yloxyacetic acid (α-TEA)], and a hydrolyzable ester derivative RRR-α-tocopheryl succinate (vitamin E succinate; VES) inhibited BALB/c mouse 66cl-4-GFP mammary tumor cell growth in vitro and in vivo. Treatment of 66cl-4-GFP cells in culture with α-TEA or VES Induced dose-dependent DNA synthesis arrest and apoptosis and inhibited colony formation. Liposomal formulations of α-TEA delivered orally or by aerosol significantly reduced subcutaneous 66cl-4-GFP tumor burden and metastasis to lung and lymph nodes. Liposomal formulations of VES delivered by aerosol significantly reduced tumor burden and lung metastasis, but not lymph node metastasis. Unlike α-TEA, VES was ineffective in reducing tumor burden and metastasis to lungs and lymph nodes when administered orally. Analyses of tumor sections showed that α-TEA delivered by either method significantly reduced tumor cell proliferation as measured by KI67, and increased apoptosis as measured by terminal deoxynucleotldyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), whereas VES delivered by aerosol reduced tumor cell proliferation and increased apoptosis, but not significantly. In summary, the nonhydrolyzable ether vitamin E derivative α-TEA was effective in reducing tumor burden and metastasis when delivered either by aerosol or orally, whereas the hydrolyzable ester vitamin E derivative VES was effective only when delivered by aerosol.

α-TEA plus cisplatin reduces human cisplatin-resistant ovarian cancer cell tumor burden and metastasis

A novel nonhydrolyzable ether-linked acetic acid analog of vitamin E, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)-chroman-6-yloxyacetic acid (α-TEA) in combination with cisplatin, reduces tumor burden of A2780/cp70 (cp70) cisplatin-resistant human ovarian cancer cells xenografted into immune compromised nude mice. Two xenograft studies were conducted using cp70 cells stably expressing green fluorescent protein (cp70-GFP) subcutaneously transplanted into NU/NU mice. For studies 1 and 2, α-TEA was formulated in liposomes and delivered by aerosol such that approximately 36 μg and 72 μg of α-TEA were deposited in the respiratory tract of each mouse each day, respectively. Cisplatin at 5 mg/kg was administered by intraperitoneal injections once weekly for the first 3 weeks in Study 1 and on the third and 10th days following treatment initiation in Study 2. The combination α-TEA + cisplatin treatment reduced tumor burden and metastasis of cp70-GFP cells in comparison to control mice or mice treated with α-TEA or cisplatin singly. A significant reduction (P < 0.001) in growth of subcutaneous transplanted tumors was obtained with α-TEA + cisplatin for both studies. Visible metastases were observed in the lungs of animals from control and cisplatin-treated groups but not in animals from the α-TEA- or α-TEA + cisplatin–treated groups. The α-TEA + cisplatin significantly reduced the total number of lung and axillary lymph node micrometastasis (P < 0.03 and P < 0.0001, respectively). Analyses of tumor sections showed the α-TEA + cisplatin treatment group, in comparison to control, to have a significantly lower level of cell proliferation (Ki-67 staining; P < 0.0001) and a significantly higher level of apoptosis (terminal deoxynucleotidyl transferase–mediated nick end labeling [TUNEL]; P < 0.0001). In summary, combinations of α-TEA + cisplatin significantly reduced tumor burden and metastases in a xenograft model of cisplatin-resistant human ovarian cancer cells. These data show promise for combination α-TEA + cisplatin chemotherapy for ovarian cancer.

α-TEA inhibits survival and enhances death pathways in cisplatin sensitive and resistant human ovarian cancer cells

RRR-α-tocopherol ether linked acetic acid analog (α-TEA), is a potential chemotherapeutic agent for ovarian cancer. Pro-death and pro-life signaling pathways were studied to understand the anti-cancer actions of α-TEA on cisplatin-sensitive (A2780S) and -resistant (A2780/cp70R) human ovarian cancer cells. Both cell lines were refractory to Fas; whereas, α-TEA sensitized them to Fas signaling. α-TEA increased levels of Fas message, protein and membrane-associated Fas. Neutralizing antibodies to Fas or Fas L partially blocked α-TEA-induced apoptosis. α-TEA induced prolonged activation of c-Jun N-terminal kinase (JNK) and its substrate c-Jun; Bax conformational change; and cleavage of Bid and caspases-8, -9 and -3. Chemical inhibitors of JNK, and caspases blocked α-TEA-induced apoptosis. α-TEA decreased phosphorylation of protein kinase B (Akt/PKB) and extracellular signal-regulated kinase (ERK1/2), as well as cellular FLICE-like inhibitory protein (c-FLIP) and Survivin protein levels. Knockdown of Akt and ERK activity using phosphoinositide- 3-kinase (PI3K) and mitogen-activated protein kinase kinase (MKK1) inhibitors enhanced α-TEA-induced apoptosis. Over-expression of constitutively active Akt2 and MKK1 blocked α-TEA-induced apoptosis. Collectively, data show α-TEA to be a potent apoptotic inducer of both cisplatin-sensitive and -resistant human ovarian cancer cells via activating death receptor Fas signaling and suppressing anti-apoptotic AKT and ERK targets.

Studies of Osteotropism on Both Sides of the Breast Cancer-Bone Interaction

Abstract:  While important advances have been made in the treatment of breast cancer (BrCa), little progress has been made in developing therapies for metastasis to bone, a complication that signals entry of the disease into an incurable phase. The process of identifying genes and gene signatures of BrCa associated with metastasis has begun. In contrast, knowledge of the contributions of bone to tumor–stroma interaction is still rudimentary. We are performing research designed to elucidate the mechanisms by which human BrCa metastasizes to bone (osteotropism). With evidence mounting that there is mutual recognition of BrCa and bone, we are investigating osteotropism from both sides of the tumor–stroma interface. We created a novel “all human” model in which human bone is transplanted into immunodeficient (NOD/SCID) mice. Human BrCa cells are injected into the mammary fat pad. Metastases later appear as metastases in the human bone, but not mouse skeleton. The model recapitulates the metastatic sequence occurring in patients. Using DNA microarrays, we plan to identify putative osteotropic genes expressed by metastatic BrCa cells. We will test the hypothesis that distinct “tool kits” are used by BrCa metastasizing to human bone. In addition, using human tissue‐engineered bone, we are identifying components within bone stroma essential for metastasis, and osteotropism genes expressed by bone in response to the presence of BrCa. We recently demonstrated that tissue‐engineered bone based on a silk sponge platform is a target for human BrCa metastasis, even in preference to the mouse skeleton.