Lung Metastatic Burden Research Articles

MTOR inhibition as an adjuvant therapy in a metastatic model of HPV+ HNSCC.

Effective treatments for recurrent/metastatic human papillomavirus-positive (HPV+) head and neck squamous cell cancer (HNSCC) are limited. To aid treatment development, we characterized a novel murine model of recurrent/metastatic HPV+ HNSCC. Further analysis of the parental tumor cell line and its four recurrent/metastatic derivatives led to preclinical testing of an effective treatment option for this otherwise fatal disease. Reverse phase protein arrays identified key signaling cascades in the parental and recurrent/metastatic cell lines. While protein expression profiles differed among the recurrent/metastatic cell lines, activated proteins associated with the mTOR signaling cascade were a commonality. Based on these data, mTOR inhibition was evaluated as an adjuvant treatment for recurrent/metastatic disease. mTOR activity and treatment response were assessed in vitro by western blot, Seahorse, proliferation, clonogenic, and migration assays. Standard-of-care cisplatin/radiation therapy (CRT) versus CRT/rapamycin were compared in vivo. Low-dose rapamycin inhibited mTOR signaling, decreasing proliferation (43%) and migration (62%) while it enhanced CRT-induced cytotoxicity (3.3 fold) in clonogenic assays. Furthermore, rapamycin re-sensitized CRT-resistant, metastatic tumors to treatment in vivo, improving long-term cures (0–30% improved to 78–100%, depending on the recurrent/metastatic cell line) and limiting lymph node metastasis (32%) and lung metastatic burden (30 fold). Studies using immune compromised mice suggested rapamycin's effect on metastasis is independent of the adaptive immune response. These data suggest a role of mTOR activation in HPV+ HNSCC recurrent/metastatic disease and that adjuvant mTOR inhibition may enhance treatment of resistant, metastatic cell populations at the primary site and limit distant metastasis.

Regulation of triple-negative breast cancer cell metastasis by the tumor-suppressor liver kinase B1.

Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), has been identified as a tumor suppressor in many cancers including breast. Low LKB1 expression has been associated with poor prognosis of breast cancer patients, and we report here a significant association between loss of LKB1 expression and reduced patient survival specifically in the basal subtype of breast cancer. Owing to the aggressive nature of the basal subtype as evidenced by high incidences of metastasis, the purpose of this study was to determine if LKB1 expression could regulate the invasive and metastatic properties of this specific breast cancer subtype. Induction of LKB1 expression in basal-like breast cancer (BLBC)/triple-negative breast cancer cell lines, MDA-MB-231 and BT-549, inhibited invasiveness in vitro and lung metastatic burden in an orthotopic xenograft model. Further analysis of BLBC cells overexpressing LKB1 by unbiased whole transcriptomics (RNA-sequencing) revealed striking regulation of metastasis-associated pathways, including cell adhesion, extracellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT). In addition, LKB1 overexpression inhibited EMT-associated genes (CDH2, Vimentin, Twist) and induced the epithelial cell marker CDH1, indicating reversal of the EMT phenotype in the MDA-MB-231 cells. We further demonstrated marked inhibition of matrix metalloproteinase 1 expression and activity via regulation of c-Jun through inhibition of p38 signaling in LKB1-expressing cells. Taken together, these data support future development of LKB1 inducing therapeutics for the suppression of invasion and metastasis of BLBC.

Abstract LB-267: Role of VEGFR-1 signaling in obesity-induced tumor progression

Abstract Background: Obesity associates with angiogenesis and increased macrophage infiltration in adipose tissues during weight gain. Whether these effects also occur in cancer to promote tumor progression in obese condition remains unclear. We have shown that the vascular endothelial growth factor receptor-1 (VEGFR-1) pathway can modulate tumor angiogenesis and recruitment of tumor-associated macrophages (TAMs). Here, we tested the emerging hypothesis that obesity enhances tumor progression and metastasis by augmenting angiogenesis and TAM recruitment via activation of the VEGFR-1 pathway. Methods: We used a high-fat diet-induced obesity model in either wild type (WT) or VEGFR-1 tyrosine kinase null (Flt1TK-/-) C57BL/6 mice. Then, we implanted orthotopically syngeneic pancreatic (PAN02) or breast (E0771) carcinomas. We evaluated the role of VEGFR-1 activity on systemic metabolism, tumor angiogenesis and immune environment, and tumor growth and metastasis. Results: Obesity increased p38-MAPK activation and TAM infiltration, tumor growth (p = 0.001) and metastasis (p = 0.035) in PAN02 tumors. VEGFR-1 inhibition reduced tumor growth (p = 0.007) and metastasis (p = 0.017) in obese but not lean mice. This was associated with a decreased p38-MAPK activity and a shift in TAM polarization towards the M1 phenotype with reduced secretion of pro-tumor cytokines, but no change in vascular density or number of TAMs. In the E0771 model, VEGFR-1 inhibition reduced MMP-9 expression and decreased lung metastatic burden (p = 0.026) in obese mice. In addition to these tumor effects, VEGFR-1 inhibition reduced weight gain, but caused metabolic disorder-hyperinsulinemia-during obesity. Combining metformin with VEGFR-1 inhibition not only prevented this metabolic alteration, but also by recruiting cytotoxic cells further decreased tumor growth in the PAN02 model (p = 0.047). Conclusion: Inactivation of VEGFR-1 signaling prevents weight gain and obesity-induced acceleration of tumor progression in pancreatic and breast cancer models. Targeting VEGFR-1 signaling axis in combination with an anti-diabetic drug such as metformin might be a considerable cancer therapeutic option in the obese setting. Note: This abstract was not presented at the meeting. Citation Format: Joao Incio, Joshua Tam, Nuh Rahbari, Priya Suboj, Daniel McManus, Shan Chin, Trupti Vardan-Kaur, Ana Batista, Suboj Babycutty, Keehoon Jung, Anna Khachatryan, Masabumi Shibuya, Raquel Soares, Dan Duda, Rakesh K. Jain, Dai Fukumura. Role of VEGFR-1 signaling in obesity-induced tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-267. doi:10.1158/1538-7445.AM2015-LB-267

Pulmonary alveolar macrophages contribute to the premetastatic niche by suppressing antitumor T cell responses in the lungs.

In contrast to tumor-associated macrophages, myeloid-derived suppressor cells, or inflammatory monocytes, functions of tissue resident macrophages, including alveolar macrophages (AM), in cancer were not well studied. Using a mouse model of breast cancer, we show that AM promote cancer metastasis to the lungs by suppressing antitumor T cells in this organ. AM accumulated in the premetastatic lungs through complement C5a receptor-mediated proliferation but not through recruitment from the circulation. AM preconditioned by breast tumors inhibited Th1 and favored generation of Th2 cells that had lower tumoricidal activity than Th1 cells. In addition, AM reduced the number and maturation of lung dendritic cells by regulating TGF-β in the lung environment. Depletion of AM reversed immunosuppression imposed by these cells and strengthened local Th1 responses, which significantly reduced lung metastatic burden. C5a receptor deficiency, which also lessens myeloid-derived suppressor cells in the premetastatic niche, synergized with the depletion of AM in preventing metastasis, leading to protection of mice from lung metastases. This study identifies AM as a new component of the premetastatic niche, which is harnessed by tumors to impose immunosuppression, and as a new target for cancer immunotherapies to eliminate or reduce metastasis. Because the lungs are the most common target for hematogenous metastasis, this research offers a plausible explanation for susceptibility of the lungs to cancer metastasis.

Abstract 2819: Efficacy and safety of the Ad-GM·CAIX dendritic cell-based vaccine in treating in vivo metastatic renal cell carcinoma compared to sunitinib monotherapy and simultaneous vaccine-sunitinib combination therapy

Abstract Metastatic clear cell renal cell carcinoma (ccRCC) disease is responsible for significant morbidity and represents the main cause of death in patients with advanced ccRCC. We have developed a novel dendritic cell based vaccine targeting human carbonic anhydrase IX (hCAIX; DC-Ad-GM·CAIX) and compared it with the ccRCC standard-of-care drug, sunitinib as a monotherapy and in simultaneous vaccine-sunitinib combination therapy. Immunocompetent mice (Balb/c) were orthotopically-transplanted with syngeneic RCC-hCAIXpositive (NPR-IX) tumor cells, immunized, and/or treated with sunitinib at low-dose (5mg/kg/d), high-dose (40mg/kg/d) or untreated. At termination, primary tumor size (weight), lung metastatic burden, hCAIX and immune-markers expression levels were compared. Mono-immunotherapy with DC-Ad-GM·CAIX vaccine suppressed metastatic tumor growth: the total number of metastatic heterotypic foci (i.e., hCAIX positive and negative foci) following vaccination decreased 2.5 fold compared with untreated mice (P<0.05). When counting only the hCAIX positive metastatic tumor cells, the decrease in the metastatic tumor burden compared to untreated mice was even more pronounced (>10 fold). Vaccination alone resulted in reduced primary tumor burden of RCC-hCAIXpositive cells to <25% of the tumor cell population, with the remaining cells lacking hCAIX expression (hCAIXnegative); there was no significant overall primary tumor reduction compared to untreated mice. In contrast, sunitinib, whether given as high-dose monotherapy or in combination with the vaccine, inhibited the primary orthotopic tumors, achieving 35% (P=0.0001) and 51% (P=1.7e-7) tumor reduction, respectively. However, sunitinib monotherapy was less effective in reducing the metastatic tumor burden compared with the vaccine. In fact, simultaneous administration of vaccine and sunitinib increased the metastatic tumor burden in heterotypic tumors composed also of hCAIXnegative cells. In conclusion, this preclinical study demonstrates that (i) the DC-Ad-GM·CAIX vaccine effectively controls both primary and metastatic hCAIXpositive tumors and forms the basis for a phase I trial in metastatic ccRCC patients, which has been initiated at UCLA (http://clinicaltrials.gov number NCT01826877), (ii) Antigen editing with loss of hCAIX is an important immune escape mechanism, (iii) This in vivo study raises caution regarding the use of DC-Ad-GM·CAIX vaccine in simultaneous combination therapies with sunitinib. Co-corresponding Authors: ASB and JR Citation Format: Edward N. Rampersaud, Jonathan W. Said, Adrian Bot, Frédéric D. Birkhäuser, Nils Kroeger, Gang Zeng, Fairooz F. Kabbinavar, Antoni Ribas, Allan J. Pantuck, Arie S. Belldegrun, Joseph Riss. Efficacy and safety of the Ad-GM·CAIX dendritic cell-based vaccine in treating in vivo metastatic renal cell carcinoma compared to sunitinib monotherapy and simultaneous vaccine-sunitinib combination therapy. [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 2819. doi:10.1158/1538-7445.AM2014-2819

Abstract 1571: The tumor suppressor Liver Kinase B1 inhibits triple-negative breast cancer cell metastasis via regulation of AP-1 signaling

Abstract The basal sub-type, which shares features with triple-negative breast cancer (TNBC), is among the most lethal breast cancer subtype, characterized by a highly aggressive and metastatic phenotype. Although pathways that may represent targets for novel therapeutic intervention for basal like breast cancer (BLBC) have begun to be elucidated, the ability to define and selectively target the invasive and metastatic phenotype of basal-type/TNBC remains a major challenge facing the breast cancer field. Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), is a known tumor suppressor in many cancers including breast. Low LKB1expression has been observed in breast cancer patients and we report a significant association between loss of LKB1 expression and poor prognosis specifically in the basal sub-type of breast cancer. Induction of LKB1 expression in BLBC cell lines inhibited invasiveness in vitro as well as lung and brain metastatic burden in an orthotopic xenograft tumor model. Further analysis of BLBC cell lines overexpressing LKB1 by next generation sequencing (RNA-seq) revealed striking regulation of metastasis-associated pathways, including cell adhesion, extra cellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT). We further demonstrated marked inhibition of matrix metalloproteinase 1 (MMP-1) expression and activity via regulation AP-1 family member cJun. Additionally, LKB1 overexpression inhibited EMT-associated genes (CDH2, Vimentin, Twist) and induced the epithelial cell marker CDH1, indicating a reversal of the EMT phenotype in a triple-negative breast cancer cell line MDA-MB-231. We have demonstrated a role for LKB1 expression in the regulation of cell invasion and metastasis in addition to tumorigenesis. Taken together these data support future development of therapeutic agents to induce the LKB1 signaling pathway in BLCB/triple-negative breast cancer. Citation Format: Lyndsay V. Rhodes, Chandra R. Tate, Hope E. Burks, Van T. Hoang, Diari Gilliam, Elizabeth C. Martin, Steven Elliott, David FB Miller, Aaron Buechlein, Douglas Rusch, Haixu Tang, Kenneth P. Nephew, Matthew E. Burow, Bridgette M. Collins-Burow. The tumor suppressor Liver Kinase B1 inhibits triple-negative breast cancer cell metastasis via regulation of AP-1 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 1571. doi:10.1158/1538-7445.AM2014-1571

Folate-targeted paclitaxel-conjugated polymeric micelles inhibits pulmonary metastatic hepatoma in experimental murine H22 metastasis models.

Hepatocellular carcinoma shows low response to most conventional chemotherapies; additionally, extrahepatic metastasis from hepatoma is considered refractory to conventional systemic chemotherapy. Target therapy is a promising strategy for advanced hepatoma; however, targeted accumulation and controlled release of therapeutic agents into the metastatic site is still a great challenge. Folic acid (FA) and paclitaxel (PTX) containing composite micelles (FA-M[PTX]) were prepared by coassembling the FA polymer conjugate and PTX polymer conjugate. The main purpose of this study is to investigate the inhibitory efficacy of FA-M(PTX) on the pulmonary metastasis of intravenously injected murine hepatoma 22 (H22) on BALB/c mice models. The lung metastatic burden of H22 were measured and tissues were analyzed by immunohistochemistry and histology (hematoxylin and eosin stain), followed by survival analysis. The results indicated that FA-M(PTX) prevented pulmonary metastasis of H22, and the efficacy was stronger than pure PTX and simple PTX-conjugated micelles. In particular, the formation of lung metastasis colonies in mice was evidently inhibited, which was paralleled with the downregulated expression of matrix metalloproteinase-2 and matrix metalloproteinase-9. Furthermore, the mice bearing pulmonary metastatic hepatoma in the FA-M(PTX) group gained significantly prolonged survival time when compared with others given equivalent doses of PTX of 30 mg/kg. The enhanced efficacy of FA-M(PTX) is theoretically ascribed to the target effect of FA; moreover, the extensive pulmonary capillary networks may play a role. In conclusion, FA-M(PTX) displayed great potential as a promising antimetastatic agent, and the FA-conjugated micelles is a preferential targeted delivery system when compared to micelles without FA.

MP35-06 EFFECTS OF DENDRITIC CELL-BASED AD-GM∙CAIX VACCINE AS MONOTHERAPY OR IN COMBINATION WITH SUNITINIB IN AN IMMUNOCOMPETENT MOUSE MODEL OF METASTATIC RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2014MP35-06 EFFECTS OF DENDRITIC CELL-BASED AD-GM∙CAIX VACCINE AS MONOTHERAPY OR IN COMBINATION WITH SUNITINIB IN AN IMMUNOCOMPETENT MOUSE MODEL OF METASTATIC RENAL CELL CARCINOMA Edward N. Rampersaud, Jonathan W. Said, Adrian Bot, Frédéric D. Birkhäuser, Nils Kroeger, Gang Zeng, Fairooz F. Kabbinavar, Antoni Ribas, Allan J. Pantuck, Arie S. Belldegrun, and Joseph Riss Edward N. RampersaudEdward N. Rampersaud More articles by this author , Jonathan W. SaidJonathan W. Said More articles by this author , Adrian BotAdrian Bot More articles by this author , Frédéric D. BirkhäuserFrédéric D. Birkhäuser More articles by this author , Nils KroegerNils Kroeger More articles by this author , Gang ZengGang Zeng More articles by this author , Fairooz F. KabbinavarFairooz F. Kabbinavar More articles by this author , Antoni RibasAntoni Ribas More articles by this author , Allan J. PantuckAllan J. Pantuck More articles by this author , Arie S. BelldegrunArie S. Belldegrun More articles by this author , and Joseph RissJoseph Riss More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1049AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Metastatic clear cell renal cell carcinoma (ccRCC) poses a therapeutic challenge because of its resistance to conventional treatment. This study evaluates the efficacy of a novel dendritic cell based vaccine targeting human carbonic anhydrase IX (hCAIX; DC-Ad-GM∙CAIX) in treating metastatic ccRCC as monotherapy and in combination with sunitinib. METHODS Balb/c mice were orthotopically-transplanted with syngeneic RCC-hCAIXpositive (NPR-IX) tumor cells, immunized, and/or treated with sunitinib at low-dose (5mg/kg/d), high-dose (40mg/kg/d) or left untreated. At termination, primary tumor size (weight), lung metastatic burden (number of foci and their size), hCAIX tumor expression and immune-markers were evaluated and compared. RESULTS Mono-immunotherapy with DC-Ad-GM∙CAIX vaccine suppressed metastatic tumor growth: the total number of metastatic heterotypic foci following vaccination decreased 2.5 fold compared with untreated mice (P<0.05). When counting only the hCAIX positive metastatic tumor cells, the decrease in the metastatic tumor burden compared to untreated mice was even more pronounced (>10 fold). Vaccination alone resulted in reduced primary tumor burden of RCC-hCAIXpositive cells to <25% of the tumor cell population, with the remaining cells lacking hCAIX expression (hCAIXnegative); there was no significant overall primary tumor reduction compared to untreated mice. In contrast, sunitinib, whether given as high-dose monotherapy or in combination with the vaccine, inhibited the primary orthotopic tumors, achieving 35% (P=0.0001) and 51% (P=1.7e-7) tumor reduction, respectively. However, sunitinib monotherapy was less effective in reducing the metastatic tumor burden compared with the vaccine. In fact, simultaneous administration of vaccine and sunitinib increased the metastatic tumor burden in heterotypic tumors composed also of hCAIXnegative cells. CONCLUSIONS This study demonstrates: (i) DC-Ad-GM∙CAIX vaccine is effective in clearing both primary and metastatic tumor cells expressing hCAIX, (ii) Antigen editing with loss of hCAIX is an important immune escape mechanism, and (iii) Sunitinib may be detrimental when given in combination with the vaccine. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e372 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Edward N. Rampersaud More articles by this author Jonathan W. Said More articles by this author Adrian Bot More articles by this author Frédéric D. Birkhäuser More articles by this author Nils Kroeger More articles by this author Gang Zeng More articles by this author Fairooz F. Kabbinavar More articles by this author Antoni Ribas More articles by this author Allan J. Pantuck More articles by this author Arie S. Belldegrun More articles by this author Joseph Riss More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Medroxiprogesterone acetate drives expansion of CD11b+Gr1high myeloid-derived suppressor cells which suppress NK cell effector functions in tumor-bearing mice (P2059)

Abstract Hormone replacement therapy with the progesterone analogue medroxyprogesterone acetate (MPA) is widely used in postmenopausal women for the treatment of endometrial conditions, and as a contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Our aim was to explore if MPA threats immunosurveillance to tumors by effecting myeloid-derived suppressor (MDSCs; CD11b+Gr1+) and NK cells in mammary tumor-bearing mice. We used the highly metastatic 4T1 breast tumor which does not express classical progesterone or glucocorticoid receptors. MPA did not affect primary tumor growth in 4T1-tumor bearing mice but promoted lung metastasis burden. This effect was accompanied by expansion of splenic CD11b+Gr1+ cells (mostly CD11b+Gr1high cells). Sorted CD11b+Gr1+ cells from MPA-treated tumor bearing mice showed a more pronounced inhibitory activity of NK cell degranulation in response to YAC-1 cells and IFN-γ production in response to cytokines than those isolated from untreated tumor-bearing mice. Also, MPA significantly increased the percentage of spleen NK cells in tumor-bearing mice with similar lung infiltration of CD11b+Gr1+ and NK cells as compared to untreated tumor-bearing mice. We conclude that in breast cancer-bearing hosts MPA promotes the accumulation of CD11b+Gr1+ which suppress NK-cell mediated anti-tumor activity potentially contributing to tumor progression and metastasis.

Early pneumothorax as a feature of response to crizotinib therapy in a patient with ALK rearranged lung adenocarcinoma

BackgroundSingle arm phase 1 and 2 studies on Crizotinib in ALK-positive patients so far have shown rapid and durable responses. Spontaneous pneumothoraces as a result of response to anti-cancer therapy are rare in oncology but have been documented in a number of tumour types including lung cancer. This includes cytotoxic chemotherapy as well as molecular targeted agents such as gefitinib and Bevacizumab. These often require chest drain insertion or surgical intervention with associated morbidity and mortality. They have also been associated with response to treatment. This is the first report we are aware of documenting pneumothorax as response to crizotinib therapy.Case presentationA 48-year-old Caucasian male presented with a Stage IV, TTF1 positive, EGFR wild-type adenocarcinoma of the lung. He received first line chemotherapy with three cycles of cisplatin-pemetrexed chemotherapy with a differential response, and then second-line erlotinib for two months before further radiological evidence of disease progression. Further analysis of his diagnostic specimen identified an ALK rearrangement by fluorescence in situ hybridization (FISH). He was commenced on crizotinib therapy 250 mg orally twice daily. At his 4-week assessment he had a chest radiograph that identified a large left-sided pneumothorax with disease response evident on the right. Chest CT confirmed a 50% left-sided pneumothorax on a background of overall disease response. A chest tube was inserted with complete resolution of the pneumothorax that did not recur following its removal.ConclusionOur case demonstrates this potential complication of crizotinib therapy and we therefore recommend that pneumothorax be considered in patients on crizotinib presenting with high lung metastatic burden and with worsening dyspnoea.

Abstract 2060: Metronomic chemotherapy (MCT): comparison of the efficacy of two combinations of drugs for the treatment of murine mammary adenocarcinomas.

Abstract Metronomic chemotherapy (MCT) refers to the chronic, equally spaced, delivery of low doses of different chemotherapeutic drugs, without extended interruptions. We have developed two combined MCT schemes for the treatment of mammary tumors: A) cyclophosphamide (Cy) 30 mg/kg/day p.o. + celecoxib (Cel) 30 mg/kg p.o. 5 times/week, and B) Cy 20 mg/kg/day p.o. + doxorubicin (Dox) 0.5 mg/kg, 3 times/week i.p. They were tested in two experimental models of mammary adenocarcinomas: I) M-406 and II) M-234p syngeneic with inbred CBi and BALB/c mice, respectively. Our present aim was to compare efficacy and toxicity of MCT combined A and B treatments in both tumor-models. There were analyzed antitumor and antimetastatic effects, survival and toxicity. The % of reduction of tumor volume of animals in the treated group with respect to control group without treatment [median (range): AI: 77.9 (50.9-89.9), AII: 84.4 (30-99.4), BI: 95.6 (57-99.6), BII: 75.5 (62.5-96.8)] didn't differ between treatments or between tumor-models (non parametric ANOVA). The % of reduction of lung metastatic burden [AI: 99.8 (97.1-99.2) AII: 99.7 (98.7-100), BI: 90.6 (35.9-96.6), BII: 90 (48.4-99.3)] was significantly different among all the groups (P&amp;lt;0.01); Dunn's post-test showed differences in AII vs BII (P&amp;lt;0.05). The analysis of survival showed higher values in treated groups, being the % of survival increase with respect to controls [AI: 56.5 (4.3-56.5) AII: 77.6 (5.3-84.2), BI: 110.9 (60-308.2), BII: 36.1 (12.4-113)] did not reach statistical significance (P=0.054). The treatments toxicity, evaluated by evolution of body weight and total leucocytes count, was similar in both tumor-models and treatments. The antitumor therapeutic efficacy was comparable in both MCT schemes and so was the toxicity generated by them. The combination Cy + Cel was superior than Cy + Dox related to antimetastatic power, suggesting its potential use at the adjuvant setting. That observation, together with the advantage that represents the oral administration of the Cy + Cel drug combination, thus leading to a better quality of life for tumor bearers, points at its election for the treatment of mammary tumors with metronomic chemotherapy. Citation Format: María J. Rico, Leandro E. Mainetti, Herman A. Perroud, Viviana R. Rozados, O. Graciela Scharovsky. Metronomic chemotherapy (MCT): comparison of the efficacy of two combinations of drugs for the treatment of murine mammary adenocarcinomas. [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 2060. doi:10.1158/1538-7445.AM2013-2060

Abstract P1-05-15: Identification of a novel Hypoxia Inducible Factor-1 regulated gene involved in breast cancer growth and metastasis

Abstract Introduction: Hypoxia is a hallmark of most solid tumors. In response to hypoxic stress tumor cells adapt by regulating survival, metabolism and angiogenesis. The heterodimeric Hypoxia-Inducible Factor (HIF)-1 transcription factor is a master regulator of this response. HIF-1alpha protein is over-expressed in ∼30% of primary breast tumors and ∼70% of metastases, which independently correlates with poor prognosis and decreased survival in patients. Moreover, conditional deletion of Hif1a in the mammary epithelium of the MMTV-polyoma middle T (MMTV-PyMT) transgenic model of breast cancer significantly delays mammary tumor initiation and lung metastasis burden. Methods: Our lab has established primary mammary tumor epithelial cells (MTECs) from late stage carcinomas originating in PyMT+; Hif1α floxed mice (FVB/Nj strain, F11). These MTECs were exposed to either Adenovirus-eta-βgal or -Cre to create wild-type (WT) and knock-out (KO) cells, respectively. PyMT MTECs are transplantable into syngeneic FVB/Nj female recipients. Deletion of HIF-1 activity reduced primary tumor growth by ∼60% and the formation of lung macrometastases originating from mammary fat pad tumors by &amp;gt;90%. Microarray profiling was conducted to identify genes differentially expressed between WT and KO cells cultured at normoxia (21% O2) or hypoxia (0.5% O2) as well as between end-stage WT and KO tumors derived from the parental cell lines. Several genes were down-regulated in both gene sets in response to deletion of Hif1a. One mRNA of particular interest, creatine kinase brain isoform (Ckb) was down-regulated in KO cells &amp;gt;100 fold and &amp;gt;2 fold in end-stage tumors. Pools of stable Ckb knockdown (KD) PyMT cells were generated via lentiviral transduction of two independent shRNA constructs (pLKO.1 shc59 or shc61, each producing &amp;gt;75% knockdown) to determine the effects of Ckb knockdown on cell proliferation, migration and metastasis. Results: No differences in cell proliferation were observed between WT and Ckb KD cells when cultured in standard culture conditions. In contrast, when cells were introduced to the mammary fat pad, initiation of palpable tumors was delayed by &amp;gt;30 days for each shRNA KD line, suggesting that CKB function may be modulated by the tumor microenvironment. A significant decrease in invasion through Matrigel was also observed for each shRNA Ckb cell line. Likewise, when cells were introduced into circulation via tail vein injection, 20% of mice injected with either shc59 or shc61 cells developed lung metastases whereas 100% of mice injected with WT cells developed metastases. Bioinformatic analyses indicate that multiple putative HIF-1 binding sites (HREs) are present in both the mouse and human CKB gene promoters; chromatin immunoprecipiation (ChIP) experiments are in progress to validate that CKB is a direct HIF-1 target gene. Conclusions: HIF-1alpha strongly promotes metastasis, the major cause of mortality in breast cancer patients. Further characterization of genes downstream of HIF-1alpha, such as CKB, that play a key role in driving invasion and tumor initiation may identify new pathways amenable to therapeutic intervention for patients with metastatic breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-05-15.

Tight Junction Proteins Claudin-3 and Claudin-4 Control Tumor Growth and Metastases

The extent of tight junction (TJ) formation is one of many factors that regulate motility, invasion, and metastasis. Claudins are required for the formation and maintenance of TJs. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are highly expressed in the majority of ovarian cancers. We report here that CLDN3 and CLDN4 each serve to constrain the growth of human 2008 cancer xenografts and limit metastatic potential. Knockdown of CLDN3 increased in vivo growth rate by 2.3-fold and knockdown of CLDN4 by 3.7-fold in the absence of significant change in in vitro growth rate. Both types of tumors exhibited increase in birth rate as measured by Ki67 staining and decrease in death rate as reflected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Knockdown of either claudin did not alter expression of other TJ protein but did reduce TJ formation as measured by transepithelial resistance and paracellular flux of dextran, enhance migration and invasion in in vitro assays, and increase lung colonization following intravenous injection. Knockdown of CLDN3 and CLDN4 increased total lung metastatic burden by 1.7-fold and 2.4-fold, respectively. Loss of either CLDN3 or CLDN4 resulted in down-regulation of E-cadherin mRNA and protein, increased inhibitory phosphorylation of glycogen synthase kinase-3β (GSK-3β), and activation of β-catenin pathway signaling as evidenced by increases in nuclear β-catenin, the dephosphorylated form of the protein, and transcriptional activity of β-catenin/T-cell factor (TCF). We conclude that both CLDN3 and CLDN4 mediate interactions with other cells in vivo that restrain growth and metastatic potential by sustaining expression of E-cadherin and limiting β-catenin signaling.

Abstract 4219: Claudin-3 and claudin-4 regulate ovarian cancer growth rate and metastatic potential through effect on integrin and β-catenin signaling

Abstract Claudins (CLDNs) are the major structural proteins of tight junctions between epithelial cells and aberrant expression of CLDNs in cancer cells is common. We discovered that constitutive knockdown of CLDN3 or CLDN4 in human ovarian cancer cells using shRNAi vectors increased their in vivo growth rate by 2.3- and 3.7-fold, respectively. In this study we examined the effect of knocking down CLDN3 and CLDN4 on the adhesion, migration and invasion and explored underlying mechanisms. Knockdown of CLDN3 or CLDN4 caused a 2.5- and 2.2-fold decrease in adhesion but increased migration by 1.4- and 1.9-fold, and invasion by 2.9- and 3.8-fold when measured in a Boyden chamber assay. To examine the effect of CLDN3 and CLDN4 knockdown on the metastatic process, we molecularly engineered the parental, CLDN3 knockdown (CLDN3KD) and CLDN4 knockdown (CLDN4KD) cells to express the DsRed fluorescent protein and then injected one million cells into the tail vein of nu/nu mice. Mice injected with either CLDN3KD or CLDN4KD cells had significantly more metastases to the lungs and other organs by day 42 compared with mice injected with the parental cells when examined under Olympus OV100 fluorescence microscope. Quantification of total lung metastatic burden in the same sets of mice by measurement of RFP fluorescence in lung homogenates showed that there was a 1.7- and 2.4-fold increase in tumor burden, respectively, in the lungs of CLDN3KD and CLDN4KD injected mice. We next asked whether the motile and invasive phenotype of the CLDN3KD and CLDN4KD cells was the result of alteration in formation of αvβ3 complex, one of the integrins that plays an important role in tumorigenicity and metastasis. Both flow cytometric analysis and deconvoluting microscopic analysis demonstrated a marked reduction in αvβ3 expression in the CLDN3KD and CLDN4KD cells. Suppression of E-cadherin expression is regarded as one of the main molecular events responsible for dysfunction in cell-cell adhesion, and the loss of E-cadherin is a well-known prerequisite for tumor cell invasion. Interestingly, we found that knockdown of CLDN3 and CLDN4 expression in the ovarian carcinoma cells was accompanied by a 55% and 95% decrease, respectively, in E-cadherin protein levels. Measurement of E-cadherin mRNA levels by qRT-PCR showed a 60% and 83% reduction in the CLDN3KD and CLDN4 cells, respectively. In association with the decrease in E-cadherin, β-catenin signaling was augmented as evidenced by a marked increase in nuclear active (de-phosphorylated) β-catenin, activation of TOPflash promoter activity and the expression of the β-catenin/TCF-regulated target genes cyclin D1 and c-myc. Our findings demonstrate that CLDN3 and CLDN4 have large effects on migration, invasion and metastasis of ovarian cancer cells and suggest that the enhanced aggressiveness is associated with diminished αvβ3 signaling and elevated β-catenin activity. 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 4219. doi:1538-7445.AM2012-4219

Blockade of Autocrine TGF-β Signaling Inhibits Stem Cell Phenotype, Survival, and Metastasis of Murine Breast Cancer Cells

Transforming growth factor beta (TGF-β) signaling has been implicated in driving tumor progression and metastasis by inducing stem cell-like features in some human cancer cell lines. In this study, we have utilized a novel murine cell line NMuMG-ST, which acquired cancer stem cell (CSC) phenotypes during spontaneous transformation of the untransformed murine mammary cell line NMuMG, to investigate the role of autocrine TGF-β signaling in regulating their survival, metastatic ability, and the maintenance of cancer stem cell characteristics. We have retrovirally transduced a dominant-negative TGF-β type II receptor (DNRII) into the NMuMG-ST cell to abrogate autocrine TGF-β signaling. The expression of DNRII reduced TGF-β sensitivity of the NMuMG-ST cells in various cell-based assays. The blockade of autocrine TGF-β signaling reduced the ability of the cell to grow anchorage-independently and to resist serum deprivation-induced apoptosis. These phenotypes were associated with reduced levels of active and phosphorylated AKT and ERK, and Gli1 expression suggesting that these pathways contribute to the growth and survival of this model system. More interestingly, the abrogation of autocrine TGF-β signaling also led to the attenuation of several features associated with mammary stem cells including epithelial-mesenchymal transition, mammosphere formation, and expression of stem cell markers. When xenografted in athymic nude mice, the DNRII cells were also found to undergo apoptosis and induced significantly lower lung metastasis burden than the control cells even though they formed similar size of xenograft tumors. Thus, our results indicate that autocrine TGF-β signaling is involved in the maintenance and survival of stem-like cell population resulting in the enhanced metastatic ability of the murine breast cancer cells.

The ADAMTS1 Protease Gene Is Required for Mammary Tumor Growth and Metastasis

A disintegrin and metalloprotease with thrombospondin motifs protein 1 (ADAMTS1) is a protease commonly up-regulated in metastatic carcinoma. Its overexpression in cancer cells promotes experimental metastasis, but whether ADAMTS1 is essential for metastatic progression is unknown. To address this question, we investigated mammary cancer progression and spontaneous metastasis in the MMTV-PyMT mouse mammary tumor model in Adamts1 knockout mice. Adamts1(-/-)/PyMT mice displayed significantly reduced mammary tumor and lung metastatic tumor burden and increased survival, compared with their wild-type and heterozygous littermates. Histological examination revealed an increased proportion of tumors with ductal carcinoma in situ and a lower proportion of high-grade invasive tumors in Adamts1(-/-)/PyMT mice, compared with Adamts1(+/+)/PyMT mice. Increased apoptosis with unaltered proliferation and vascular density in the Adamts1(-/-)/PyMT tumors suggested that reduced cell survival accounts for the lower tumor burden in ADAMTS1-deficient mice. Furthermore, Adamts1(-/-) tumor stroma had significantly lesser amounts of proteolytically cleaved versican and increased numbers of CD45(+) leukocytes. Characterization of immune cell gene expression indicated that cytotoxic cell activation was increased in Adamts1(-/-) tumors, compared with Adamts1(+/+) tumors. This finding is supported by significantly elevated IL-12(+) cell numbers in Adamts1(-/-) tumors. Thus, in vivo ADAMTS1 may promote mammary tumor growth and progression to metastasis in the PyMT model and is a potential therapeutic target to prevent metastatic breast cancer.

Abstract LB-202: Therapeutic efficacy of coencapsulated cisplatin and arsenic trioxide nanobins in murine models of breast cancer

Abstract We have developed a novel strategy to encapsulate a combination of cisplatin and arsenic trioxide as a precipitate inside 100 nm liposomal nanobins, NB(Pt, As). Cisplatin is a widely used chemotherapeutic but has significant dose-limiting toxicity. Arsenic trioxide (ATO) is a potent FDA-approved therapy for acute promyelocytic leukemia; however, ATO has not been effective in clinical trials of solid tumors. Encapsulation in nanoliposomes can improve the anti-cancer efficacy of chemotherapeutic agents by extending serum half-life, increasing tumor drug delivery and attenuating toxicity. We have tested the therapeutic efficacy of NB(Pt, As) in orthotopic xenograft models of triple negative breast cancer using MDA-MB-231 and MDA-MB-435/LvBr1 cancer cell lines. Treatment with either NB(Pt, As) or cisplatin for three weeks inhibited growth of MDA-MB-231 mammary tumors in vivo. However, the NB(Pt, As) did not cause weight loss in treated animals and did not result in abnormal serum chemistries. NB(Pt, As) was also evaluated for its ability to inhibit lung metastases. Mice inoculated intraductally with highly metastatic MDA-MB-435/LvBr1 cancer cells develop mammary tumors that metastasize and form macroscopic lesions in the lung. Treatment with NB(Pt, As) was initiated when primary tumors reached∼250 mm3. Both NB(Pt, As) and cisplatin inhibited primary tumor growth in this model, whereas only NB(Pt, As) treatment significantly reduced lung metastatic burden. Our results suggest that nanobin encapsulated cisplatin and ATO is a promising nanoscale therapeutic agent for metastatic breast cancer. 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 LB-202. doi:10.1158/1538-7445.AM2011-LB-202

Abstract 1358: Focal adhesion kinase (FAK) regulates myeloid lineage cell activities that control murine tumor progression

Abstract The tumor microenvironment is a critical regulator of tumor progression. Results from a broad spectrum of experimental models and analyses of human tumor specimens provide strong evidence that cells of the myeloid lineage have functional attributes that can potentiate the growth, invasion, and metastasis of cancer. Our approach to investigate the role of the microenvironment in tumor progression is centered on the activity of focal adhesion kinase (FAK) in myeloid lineage cells. We have recently shown in macrophages that FAK, a non-receptor tyrosine kinase, regulates adhesion dynamics and migration toward soluble growth factors and chemokines located at sites of tumor growth. Additionally, mice that have FAK conditionally deleted in this lineage display reduced peritoneal infiltration of macrophages following thioglycollate-induced inflammation. These data suggest that FAK may be involved in regulating the migration of myeloid lineage cells to the tumor and the response of macrophages to factors within the tumor microenvironment. We hypothesize that FAK regulates critical aspects of myeloid/macrophage behavior that allow these cells to support tumor progression and metastasis. This hypothesis is being tested in several tumor models. First, using myeloid-specific conditional FAK knockout mice (FAK-/-), we have tested this hypothesis in the context of both a xenograft tumor model using MDA-MB-231 breast cancer cells and a spontaneous breast tumor model driven by mammary-specific expression of the polyoma virus middle T antigen (MMTV-PyVmT). Significantly fewer tumors developed from orthotopic injection of MDA-MB-231 cells in mice lacking FAK in myeloid lineage cells. F4/80 staining of tumor sections from these mice showed reduced infiltration of macrophages into the tumor mass compared to tumors from wild type (WT) mice. Initial data from the spontaneous MMTV-PyVmT tumor model show no significant differences in time to tumor onset and rate of tumor outgrowth between the FAK-/- and WT mice. Analysis of the lung metastatic tumor burden is ongoing. Second, we have conducted preliminary experiments testing the influence of FAK expression in myeloid lineage cells on the adaptive immune response to tumor growth. Following subcutaneous implantation of the syngeneic B16/F1 melanoma cell line, we observed a significant delay in the onset of tumor formation in FAK-/- mice compared to WT mice. However, when a similar study was performed in a severe combined immunodeficient (SCID) background, tumor outgrowth was identical in WT and conditional knockout mice. Based on these results, we are currently investigating mechanisms through which FAK expression in myeloid lineage cells may regulate the response of lymphocytes to the early stages of tumor outgrowth. 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 1358.

GATA3 inhibits breast cancer growth and pulmonary breast cancer metastasis

The loss of expression of the transcription factor GATA3 in breast tumors has been linked to aggressive tumor development and poor patient survival. In the present work, we address potential roles for GATA3 in breast tumor lung metastasis and progression. Using an aggressive breast cancer cell line, which metastasizes specifically to the lung, we show that GATA3 expression results in reduced tumor outgrowth in the mammary fat pad and lower lung metastatic burden in nude mice. Specifically, GATA3 expression inhibits breast cancer cell expansion inside the lung parenchyma. This phenotype correlates with the ability of GATA3 to negatively regulate the expression of several genes that promote breast cancer lung metastasis (ID1/-3, KRTHB1, LY6E and RARRES3). Conversely, the expression of genes encoding known inhibitors of lung metastasis (DLC1 (deleted in liver cancer 1) and PAEP (progestagen-associated endometrial protein)) is upregulated by GATA3. These data correlate with microarray data from human breast cancer patients, showing a strong correlation between high GATA3 expression and absence of metastases specifically to the lungs. We conclude that GATA3 inhibits primary breast tumor outgrowth and reduces lung metastatic burden by regulating key genes involved in metastatic breast tumor progression.

Wilms tumor: a new method for quantification of lung metastatic tumor burden

Wilms tumor: a new method for quantification of lung metastatic tumor burden