Growth Of Established Tumors Research Articles

Inherited Loss of Samsn1 Contributes to Increased Risk of MGUS and MM through Effects on Multiple Cell Types, Including B-Cells, Transformed Myeloma Cells, and Macrophages

Multiple myeloma (MM) is a malignancy of plasma B-cells that is invariably preceded by monoclonal gammopathy of undetermined significance (MGUS), a pre-neoplastic plasma cell proliferative disorder. C57Bl/KaLwRij (KaLwRij) is a spontaneous inbred mouse strain predisposed to benign idiopathic paraproteinemia (BIP), a plasma cell dyscrasia that parallels many features of human pathology including progression to myeloma. In both humans and mice, the genetic basis for MGUS/MM risk is unknown. We sought to identify the genetic basis for MGUS/MM risk in KaLwRij mice to shed light on genes or pathways that mediate disease risk in humans. Using an integrative approach, we combined KaLwRij x B6 haplotype analysis (418 genes) and a MM patient association study (180 genes) to identify five candidate genes likely to contribute to both murine BIP-susceptibility and human MM risk. Surprisingly, we found KaLwRij mice had homozygous germline deletion of one of the five candidate genes, Samsn1, a putative negative regulator of B-cell activation. Consistent with the reported inhibitory function of SAMSN1 in B-cells, KaLwRij mice had enhanced naïve B-cell response in vitro following stimulation with IL4 and LPS and in vivo following immunization. We found that re-expression of Samsn1 in KaLwRij-derived myeloma cell line 5TGM1 decreased MM tumor growth in vitro, suggesting that loss of Samsn1 contributes to tumor growth in part through a cell intrinsic mechanism. Importantly, germline risk alleles may also influence cell types beyond the pre-malignant B-cells. It is established that the host environment also plays a critical role in supporting myeloma growth in KaLwRij mice. We found that Samsn1 is highly expressed in macrophages, but not in other MM-supportive cell types including osteoclasts and bone marrow stromal cells. Myeloma-associated macrophages play critical roles in supporting tumor growth and are broadly classified as anti-tumor M1 macrophages and tumor-promoting M2 macrophages. Microarray analysis of macrophages from B6 and KaLwRij mice prior to onset of BIP revealed three differentially expressed genes, but only one, Samsn1, is conserved in the human genome. We found that both unpolararized primary peritoneal and MCSF cultured bone marrow macrophages from healthy KaLwRij mice had markedly elevated expression of pro-tumorigenic M2 macrophage markers FIZZ1 and YM1 compared to B6 controls, despite the absence of activating cytokines. To confirm that loss of Samsn1 influenced macrophage activation and M2 polarization, we obtained bone marrow macrophages from mice with a targeted deletion of Samsn1 and found that unpolarized Samsn1-/- cells had similarly increased pro-tumor M2 macrophage marker expression. To evaluate the biologic significance of Samsn1 loss in macrophages on MM tumor growth, we established 5TGM1 MM tumors and injected M2 polarized macrophages from B6, KaLwRij, or Samsn1-/- mice. We found that MM tumor burden was markedly enhanced by the addition of a Samsn1-/- or KaLwRij macrophages compared to wild type macrophages. Thus, we found that loss of Samsn1 in macrophages enhances M2 polarization, prior to MGUS/MM development and can promote established myeloma tumor growth. Together, this data suggests that Samsn1 is a gene contributing to BIP-susceptibility in KaLwRij mice through mutually exclusive functions in multiple cell types including B-cells and macrophages. It is likely that SAMSN1 and its pathway members contribute to human MM progression, and may represent a point for preventative therapeutic intervention. DisclosuresNo relevant conflicts of interest to declare.

Thrombin Drives Colorectal Cancer Pathogenesis at the Level of Primary Tumor Growth, Tumor Invasion and Metastasis

Cell-associated and circulating hemostatic factors have been identified as powerful determinants of tumor cell metastatis, primarily by supporting the early survival of newly formed micrometastases. However, despite substantial evidence that procoagulants and related signaling molecules can control cell proliferation, angiogenesis and other reparative processes, multiple prior studies unexpectedly demonstrated that hemostatic factors were often dispensable for the growth of established tumors. Here, we report that colorectal cancer represents at least one important exception where hemostatic factors control multiple aspects of cancer pathogenesis. Consistent with clinical findings that patients with prothrombotic mutations (homozygosity for the fVLeiden) have a ~6-fold increased risk of developing colorectal cancer, we have shown previously that mice with even a relatively modest 50% genetically-imposed diminution in circulating prothrombin exhibit a dramatically decreased tumor incidence in a murine model of inflammation-driven colon cancer. In order to determine if thrombin supports the growth of established colorectal cancer, we compared the subcutaneous growth of an aggressive C57Bl/6-derived colonic adenocarcinoma (MC38) in cohorts of wildtype mice and fIILox/- mice carrying just ~10% the normal level of circulating prothrombin. Colon cancer growth was significantly diminished in fIILox/- mice relative to control animals, resulting in a ~3-fold difference in tumor mass between cohorts at the end of the 3 week study period. Complementary studies in which hepatic prothrombin expression and circulating prothrombin levels were diminished using a specific prothrombin antisense oligonucleotide “gapmer” resulted in a similar reduction in colorectal cancer primary tumor growth. Furthermore, prothrombin “gapmer” treatment also slowed the growth of an aggressive, human-derived colorectal adenocarcinoma cell line transplanted into immunodeficient nude mice. Detailed histological analyses revealed no significant prothrombin-dependent differences in angiogenesis, tumor cell apoptosis or the numbers of tumor-associated macrophages. However, histological analyses of colonic adenocarcinoma tumors revealed a significantly lower mitotic index as well as significantly less migration of tumor cells through the panniculus muscle in tumors harvested from fIILox/- mice relative to control mice, suggesting that thrombin promotes tumor cell proliferation and local migration through tissue barriers. Complementary studies of pulmonary metastatic foci formed following intravenous injection of MC38 cells into fIILox/- and control mice further revealed that lowering prothrombin essentially eliminated pulmonary metastases, whereas numerous pulmonary foci were present in animals with normal prothrombin levels. To further characterize the role of thrombin in colorectal cancer growth, we analyzed the potential of a-thrombin to promote MC38 proliferation and invasion in vitro. Here, brief exposure to a-thrombin induced the proliferation of MC38 cells and acted as a chemoattractant when added to the lower chamber in a Boyden chamber assay. Together, these findings suggest thrombin significantly contributes to multiple stages of colon cancer pathogenesis, including tumor growth, invasion, and metastatic seeding, and emphasize the potential utility of targeting prothrombin, thrombin generation or thrombin activity as novel adjunct therapeutic strategies to impede the growth and progression of colorectal carcinoma. DisclosuresNo relevant conflicts of interest to declare.

Depletion of tumor-associated macrophages enhances the anti-tumor immunity induced by a Toll-like receptor agonist-conjugated peptide

It has been reported that lipopeptides can be used to elicit cytotoxic T lymphocyte (CTL) responses against viral diseases and cancer. In our previous study, we determined that mono-palmitoylated peptides can enhance anti-tumor responses in the absence of adjuvant activity. To investigate whether di-palmitoylated peptides with TLR2 agonist activity are able to induce anti-tumor immunity, we synthesized a di-palmitic acid-conjugated long peptide that contains a murine CTL epitope of HPV E749–57 (Pam2IDG). Pam2IDG stimulated the maturation of bone marrow-derived dendritic cells (BMDCs) through TLR2/6. After immunization, Pam2IDG induced higher levels of T cell responses than those obtained with its non-lipidated counterpart (IDG). In the prophylactic model, Pam2IDG immunization completely inhibited tumor growth, whereas IDG immunization was unable to inhibit tumor growth. However, Pam2IDG immunization could not effectively inhibit the growth of established tumors. Therefore, we further investigated whether the depletion of immunosuppressive factors could improve the therapeutic effects of Pam2IDG. Our data indicate that treatment with Pam2IDG combined with clodronate/liposome delays tumor growth and increases the survival rate. We also observed that the therapeutic effects of Pam2IDG are improved by diminishing the function of tumor-associate macrophages (TAMs) and through the use of an IL10 receptor blocking antibody or a Cyclooxygenase 2 (Cox-2) inhibitor. In conclusion, the depletion of TAMs may enhance the anti-tumor immunity of a TLR2 agonist-conjugated peptide.

MTOR inhibitors induce cell-cycle arrest and inhibit tumor growth in Epstein-Barr virus-associated T and natural killer cell lymphoma cells.

Epstein-Barr virus (EBV) infects B cells, as well as T cells and natural killer (NK) cells, and is associated with T or NK cell lymphoid malignancies. In various tumor cells, mTOR performs an essential function together with Akt with regard to cell growth. We investigated the effects of mTOR inhibitors on EBV-associated T- and NK-cell lymphomas. We investigated the Akt/mTOR activation pathway in EBV-positive and -negative T- and NK-cell lines (SNT13, SNT16, Jurkat, SNK6, KAI3, and KHYG1). We evaluated the antitumor effects of mTOR inhibitors (rapamycin and its analogue, CCI-779) against these cell lines in culture and in a murine xenograft model that was established by subcutaneous injection of SNK6 cells into NOG mice. All EBV-positive and -negative T- and NK-cell lines tested displayed activation of the Akt/mTOR pathway, and treatment with mTOR inhibitors suppressed mTOR activation. The inhibitors induced G1 cell-cycle arrest and inhibited cell proliferation in T- and NK-cell lines. Overall, T cell lines were more sensitive to rapamycin, but there were no significant differences between EBV-positive and -negative cell lines. Treatment with rapamycin did not affect lytic or latent EBV gene expression. Intraperitoneal treatment with CCI-779 significantly inhibited the growth of established tumors in NOG mice and reduced the EBV load in peripheral blood. These results suggest that inhibition of mTOR signaling is a promising new strategy for improving treatment of EBV-associated T- and NK-cell lymphoma.

SKLB316, a novel small-molecule inhibitor of cell-cycle progression, induces G2/M phase arrest and apoptosis in vitro and inhibits tumor growth in vivo

Benzothiazole derivatives have received considerable attentions for their potencies in cancer therapy. In the present study, we reported that SKLB316, a novel synthesized benzothiazole derivative, exhibits activities to inhibit colorectal and pancreatic cancer in vitro and in vivo by inducing G2/M cell cycle arrest and apoptosis. In vitro, it exhibited significant anti-proliferative activities against human cancer cells derived from different histotypes including the colorectal cancer cell line HCT116 and pancreatic cancer cell line CFPAC-1. We chose these cell lines to study the possible anti-tumor mechanism because they are sensitive to SKLB316 treatment. Flow cytometry assays showed that SKLB316 could induce G2/M cell cycle arrest. Mechanistically, SKLB316 could decrease the activities of cdc2/cyclin B1 complex, including decreasing the synthesis of cyclin B1, cdc2 and cdc25c, while accumulating the levels of phosphorylated cdc2 (Tyr15) and checkpoint kinase 2. SKLB316 could also decrease the level of cyclin E and A2. Moreover, SKLB316 could induce cancer cell apoptosis, which was associated with activation of caspase 9, downregulation of Bcl-2 and upregulation of Bax. SKLB316 could also decrease the mitochondrial membrane potential and induce the generation of reactive oxygen species in cells. The results implied that SKLB316 may induce apoptosis via the mitochondria-mediated apoptotic pathway. Moreover, SKLB316 could suppress the growth of established colorectal and pancreatic cancer tumors in nude mice without causing obvious side effects. TUNEL assays confirmed that SKLB316 could also induce tumor cell apoptosis in vivo. Taken together, these findings demonstrate the potential value of SKLB316 as a novel anti-tumor drug candidate.

Abstract 2289: Effects of TRAIL and ellagic acid on malignant melanoma A375 in vitro and in vivo

Abstract Cancer is a widespread disease characterized by unregulated cell growth and evasion of apoptotic stimuli. Most conventional therapies lack cancer cell specificity and induce cell death indirectly through cellular damage in a p53-dependent manner. A direct and selective p53-independent cancer therapy is the application of Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL). Recombinant human TRAIL (rhTRAIL) can induce apoptosis in a broad range of transformed human cells while showing minimal toxicity towards non-malignant cells. However, some cancers are resistant to rhTRAIL-induced apoptosis as a direct result of overexpressed antiapoptotic proteins. One key driver mediating the expression of such antiapoptotic proteins is Casein Kinase 2 (CK2). CK2 is upregulated in the nucleus of all cancer cells and promotes the expression of inhibitors of apoptosis resulting in resistance to cell death. Therefore, by inhibiting CK2, TRAIL-resistant cells may become more sensitive to TRAIL-induced apoptosis. We utilized cotreatment with Ellagic Acid (EA), a natural CK2 inhibitor, and TRAIL against the malignant melanoma cell line A375, which is resistant to TRAIL. Our in vitro analysis showed that TRAIL and EA, as single-agents, can inhibit cell growth in a dose-dependent manner, and the combination treatment of TRAIL plus EA synergistically enhanced the cytostatic effects of the single-agent treatments. We show that TRAIL reduces the viability of A375 cells; whereas, EA although it can inhibit growth, cannot reduce the viability of A375 as determined by Trypan Blue exclusion assays. In combination treatments, the cytotoxic effects of TRAIL were not enhanced. Annexin-V Apoptosis assays and Western Blot analysis show that TRAIL can induce apoptosis in A375 cells characterized by the formation of Annexin-V positive cells, PARP fragmentation and the activation of caspase 3. Additionally, TRAIL initiated both the extrinsic and intrinsic pathways of apoptosis marked by caspase 8 activation and the mitochondrial release of cytochrome c, respectively. EA treatment alone did not induce apoptosis and did not induce formation of Annexin-V positive cells or PARP fragmentation. Combination treatments show that EA does not potentiate TRAIL-induced apoptosis. In vivo, TRAIL-treated nude mice bearing A375 xenografts exhibited ∼80% tumor growth inhibition and a prolonged survival rate compared to the untreated control group. EA was not able to inhibit the growth of established A375 tumors and did not augment the in vivo antiproliferative effects of TRAIL. These data demonstrate that EA can enhance the cytostatic but not the cytotoxic or proapoptotic effects of TRAIL in vitro. In vivo, TRAIL can inhibit the growth of established tumors as a single-agent but is not augmented by cotreatment with EA. Citation Format: Katherine Turner, Daniel Lindner, Michael Kalafatis. Effects of TRAIL and ellagic acid on malignant melanoma A375 in vitro and in vivo. [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 2289. doi:10.1158/1538-7445.AM2014-2289

Abstract 890: Serum inflammatory biomarkers predict esophageal and lung cancer risk two years prior to diagnosis in a prospective cohort

Abstract Background: Cancer-associated inflammation drives the progression, growth, and metastasis of many established tumors, including those of the head & neck, esophagus, and lung. Limited evidence from non-aerodigestive cancers suggests that quantifiable immunologic dysfunction may precede the emergence of some clinically detectable tumors. The Golestan region of Iran provides a unique opportunity to further study the relationship between chronic inflammation and aerodigestive cancer risk, as it features a very high incidence of esophageal squamous cell carcinoma despite remarkably low prevalence of typical confounders, including tobacco and alcohol use. This study tests the hypothesis that a comprehensive panel of cancer-associated serum inflammatory biomarkers can accurately predict risk of head & neck, esophageal, and lung cancers in a prospective, high-risk cohort. Methods: This is a nested case-control pilot study of subjects enrolled in the Golestan Cohort Study, an ongoing epidemiologic project assessing cancer trends in Golestan, Iran. We measured a broad panel of cytokines, chemokines, and inflammatory molecules using Luminex® technology in serum samples collected two or more years before cancer diagnosis in 78 aerodigestive cancer cases and 81 controls. Data was analyzed using Wilcoxon rank-sum test, ROC areas of discrimination, and multivariate analysis. Results: Mean age of 159 study subjects was 60 years. Median levels (pg/ml) of several biomarkers were profoundly elevated in future esophageal and lung cancer patients, respectively, as compared to controls, including FGF-2 (341.9 and 306.7 versus 139.6, p<0.0001), GM-CSF (63.7 and 63.3 versus 21.3, p<0.0001), IFN-α2 (245.3 and 251 versus 67.6, p<0.0001), and IL-1Rα (415 and 413.2 versus 116.2, p<0.0001). Receiver operating characteristic (ROC) areas demonstrated a strong ability to distinguish esophageal and lung cancer cases from controls for FGF-2, GM-CSF, IFN-α2, IL-1Rα, and TNF-β. The highest ROC area was 88% for esophageal cancer (GM-CSF and IFN-α2) and 85% for lung cancers (GM-CSF). The same trend was not observed among head and neck cancer cases, as no biomarker had a ROC area >69% as compared to controls. Among data-linked controls, biomarker levels tended to be higher with older age, Turkmen ethnicity, and cigarette smoking, but these correlations were non-significant. Conclusions: This study provides the first direct evidence that multiple inflammatory biomarkers are coordinately elevated in future lung and esophageal cancer patients at least two years before cancer diagnosis, suggesting that baseline serum levels of certain cancer-associated biomarkers may represent a useful adjunctive screening measure to determine aerodigestive cancer risk. Citation Format: Brieze R. Keeley, Farhad Islami, Akram Pourshams, Hossein Poustchi, Jamie S. Pak, Paul Brennan, Hooman Khademi, Shu-hsia Chen, Eric M. Genden, Christian C. Abnet, Sanford M. Dawsey, Paolo Boffetta, Reza Malekzadeh, Andrew G. Sikora. Serum inflammatory biomarkers predict esophageal and lung cancer risk two years prior to diagnosis in a prospective cohort. [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 890. doi:10.1158/1538-7445.AM2014-890

Abstract 2690: Sorafenib/regorafenib and phosphatidyl inositol 3 kinase/thymoma viral proto-oncogene inhibition interact to kill tumor cells

Abstract The present study aimed to determine whether the multikinase inhibitors, sorafenib/regorafenib, worked in conjunction with PI3K/AKT inhibitors to enhance tumor cell death. It has been noted that combination of sorafenib/regorafenib with a PI3K inhibitor acetic acid (PX-866) resulted in tumor cell death, in a greater than additive fashion, in liver, colorectal, lung, breast, kidney and brain cancer cells. Similar data was obtained using AKT inhibitors perifosine and MK2206. Furthermore, even cells lacking PTEN remained as sensitive to this combinational approach as cells expressing PTEN. PX-866 treatment abolished AKT/GSK3 phosphorylation, with tumor cell death correlating with reduced activity of AKT and mTOR. Expression of activated AKT and to a lesser extent activated mTOR reduced drug combination lethality. Expression of B-cell lymphoma-extra large or dominant negative caspase 9, but not cellular FLICE-inhibitory protein short, protected cells from the drug combination. Treatment of cells with PX-866 increased protein levels of p62, LAMP2 and LC3/LC3II which correlated with the resulting increase in LC3-GFP vesicle numbers. Exposure of PX-866 treated cells to sorafenib reduced p62 and LAMP2 levels, decreased the ratio of LC3 to LC3II and reduced LC3-GFP vesicle levels. Knockdown of Beclin1 or ATG5 suppressed drug toxicity by ∼40%. In vivo, combination of either sorafenib and PX-866 or regorafenib and MK2206 cooperated to suppress the growth of established HuH7 and HCT116 tumors, respectively. Collectively our data demonstrates that the combination of sorafenib family kinase inhibitors with inhibitors of the PI3K/AKT pathway kills tumor cells in vitro and in vivo. Citation Format: Larry A. Booth, Nichola A. Cruickshanks, G B. Sajithlal, Hossein A. Hamed, Seyedmehrad Tavallai, J Syed, Steven Grant, Andrew Poklepovic, Paul Dent. Sorafenib/regorafenib and phosphatidyl inositol 3 kinase/thymoma viral proto-oncogene inhibition interact to kill tumor cells. [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 2690. doi:10.1158/1538-7445.AM2014-2690

Abstract 4759: Integral role of the PI3K-AKT-mTOR pathway in DDR-mediated antitumor actions of PARP inhibitor in triple-negative breast carcinogenesis

Abstract Background: The PI3K pathway, in addition to its pro-proliferative and anti-apoptotic effects on tumor cells, is known to contribute to DNA-damage repair (DDR). We hypothesized that GDC-0980, a dual PI3K-mTOR inhibitor, would potentiate an anti-tumor effect in BRCA-competent TNBC cells when combined with the PARP inhibitor, ABT888 and carboplatin. Methods: Athymic mice bearing BRCA-competent TNBC xenograft tumors were treated with GDC-0980 alone or in combination with ABT888 and carboplatin. Mechanism-based in vitro studies (MTT, CelltiterGLO, flow cytometry-based live/dead cell, cell cycle analyses, anchorage independent soft-agar, anchorage dependent 3D ON-TOP, apoptosis, cell signaling markers assays) using a panel of 5-7 BRCA-wt /mutant TNBC cell lines were performed. Results: GDC-0980 treatment led to DNA damage (increased pgH2AX; WB, IF), gain in PAR and a subsequent sensitization of BRCA-competent TNBC cells to ABT888 plus carboplatin in a time-dependent manner. The treatment (1) decreased proliferation signals (pAKT, pP70S6K, p4EBP1, pS6RP), PAR/PARP, PAR/pgH2AX, live/dead cell ratios, cell cycle progression and clonogenic 3D growth, and (2) increased apoptosis markers (cl-caspase3, 9, BIM, cl-PARP, and annexinV positivity). These effects were more pronounced in PTEN-null MDA-MB468 than in RAS/RAF mutated MDA-MB231 cells. Three-dimensional projection movie showed that (1) GDC-0980 alone attenuated nuclear pγH2AXS139 foci in MDA-MB468 cells at 24 hours and (2) cytoplasmic cl-caspase3 increased in GDC-0980+ABT888+carboplatin treated MDA-MB468 cells at 72 hours. Finally, GDC-0980 in combination with ABT888 plus carboplatin blocked the growth of established xenograft tumors by 80-90% with a concomitant decrease in tumor Ki67, cl-caspse3, pVEGFR, CD31, p4EBP1, and pS6RP IHC-levels. Conclusion: This is the first mechanism-based study to demonstrate the inhibition of DDR as another mode of action of GDC-0980. Our data revealed that in a BRCA-competent model, GDC-0980 enhanced the antitumor efficacy of ABT888 in the presence of carboplatin by inhibiting the DDR system in conjunction with inhibition of the PI3K-mTOR pathway mediated pro-proliferative and anti-apoptotic signals. Citation Format: Brian Leyland-Jones, Jennifer Carlson, Yuliang Sun, Lori Friedman, Pradip De, Nandini Dey. Integral role of the PI3K-AKT-mTOR pathway in DDR-mediated antitumor actions of PARP inhibitor in triple-negative breast carcinogenesis. [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 4759. doi:10.1158/1538-7445.AM2014-4759

Abstract SY43-03: Screening for triple negative breast cancer vulnerabilities

Abstract There is no targeted therapy for triple negative breast cancers (TNBC), which have the worst prognosis of human breast cancers. TNBCs are prone to relapse and metastasize after cytotoxic drug treatment. This group of cancers, defined by low or absent expression of estrogen, progesterone and Her2 receptors, are heterogeneous in genetic, epigenetic and phenotypic features, making it difficult to identify specific drug targets suitable for this group of cancers as a whole. About half or more of TNBCs have an epithelial phenotype (classified based on gene expression profiling as basal-like or basal-A) and a large minority of the remaining tumors are mesenchymal (classified as basal-B). To identify selective genetic dependencies of the basal-like subtype of TNBC that are not required for survival of normal breast epithelial cells, which might be good targets for targeted drug therapy, we first performed a genome-wide siRNA lethality screen that compared two human breast epithelial TNBC cell lines transformed with the same genes in different media - basal-like BPLER and myoepithelial HMLER. BPLER are highly malignant and enriched for tumor-initiating cells, forming tumors in nude mice after injection of just 50 cells, while the more differentiated HMLER cell line only forms tumors when more than 50,000 cells are injected. The screen identified 154 genes on which BPLER, but not HMLER, depended. Expression of the 154 BPLER dependency genes correlated with poor prognosis in breast, but not lung or colon, cancer. Of note, no oncogenic kinases, which are often chosen as cancer drug targets, were identified in the screen. Proteasome genes were overrepresented hits. Basal-like TNBC lines as a group were selectively sensitive to proteasome inhibitor drugs relative to normal epithelial, luminal breast cancer and mesenchymal TNBC lines. Proteasome inhibition reduced growth of established basal-like TNBC tumors in mice and blocked tumor-initiation and metastasis. Tumor-initiating cells (T-ICs) within basal-like cancers did not survive proteasome inhibition. Developing ways to target T-ICs is a priority of cancer drug development. Proteasome addiction in basal-like TNBCs was mediated by NOXA and linked to MCL-1 dependence, which was also a common dependency gene of basal-like TNBCs. Because of the heterogeneity of TNBCs, we next assessed how many of the BPLER dependencies were shared with other breast cancers. A targeted screen was performed comparing the viability of 10 breast cancer cell lines (3 luminal A, 5 basal-A and 2 basal-B TNBCs) and telomerase-immortalized normal breast epithelial cells after knockdown of the 154 BPLER dependency genes. This screen confirmed the selective dependence of basal-A TNBCs on the proteasome and MCL-1. This screen identified 17 additional hits that were shared dependencies in at least 4 of 5 basal-like TNBC cell lines. A novel hit, required for survival of 4 of 5 basal-A TNBC cell lines, but not normal breast epithelial cells, was the spindle checkpoint regulator NDC80. This work was supported by Department of Defense grants to FP and JL and by a grant from the Harvard Stem Cell Institute. The authors have no financial conflicts of interest to disclose. Citation Format: Fabio Petrocca, Gabriel Altschuler, Shen Mynn Tan, Marc L. Mendillo, Haoheng Yan, D. Joseph Jerry, Andrew L. Kung, Winston Hide, Tan A. Ince, Judy Lieberman. Screening for triple negative breast cancer vulnerabilities. [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 SY43-03. doi:10.1158/1538-7445.AM2014-SY43-03

Abstract 1816: Assessment of anti-tumor activity of the cathepsin L inhibitor, KGP94

Abstract Purpose: The cysteine cathepsin proteases are known to play an important role in tumor progression, invasion and angiogenesis. The present study evaluated the anti-tumor activity and pharmacokinetics of the small-molecule cathepsin L inhibitor KGP94. Materials and Methods: Male CDF1 or C3H/HeNHsd mice were inoculated on the right rear foot with a C3H mammary carcinoma or a SCCVII carcinoma, respectively. A solution of KGP94 was prepared each day by dissolving in a mixture of 10% Tween 80 and 90% HEPES-buffer. It was intraperitoneally injected at 0.01 ml/g mouse bodyweight. Various doses (1-20 mg/kg) were administered between 1-20 days, starting from the day of tumor inoculation. Anti-tumor activity was assessed by determining the tumor growth time, which was the time in days to reach a volume of 500 mm3 (TGT500). Pharmacokinetics of KGP94 were investigated by drawing blood samples before, and up to 24 hours after KGP94 injection. Results are listed as Mean (± Standard Error). One-way ANOVA comparison of group means was performed, and a P<0.05 was considered significant. Results: The TGT500 for control animals was 18.0 days (± 0.3) for the C3H mammary carcinoma and 13.6 days (± 0.7) for the SCCVII carcinoma. Treating tumors with KGP94 significantly increased the TGT500 for both tumor models to around 21 and 17 days respectively, when doses were at 10.0 mg/kg or higher. At a dose of 5.0 mg/kg the TGT500 of the C3H mammary carcinoma was significantly increased to 21.4 days (± 1.1), whereas the TGT500 of the SCCVII carcinoma was unchanged at 13.8 (± 0.2). At lower KGP94 doses neither tumor model showed significant growth delay. Additional experiments with the C3H mammary carcinoma model showed that treatment was most effective when started immediately after tumor inoculation, and that treatment for 5 days was just as effective as 20 days. Pharmacokinetic analysis of KGP94 levels in mouse plasma is ongoing. Conclusion: In the C3H mammary carcinoma KGP94 significantly delayed tumor growth, but the effect was primarily on the early phase following tumor cell inoculation. This suggests that KGP94 may have a more significant role in inhibiting the establishment of metastasis rather than influencing the growth of established tumors. Significant growth delay was also observed in the SCCVII tumor model. However, it occurred with higher doses of KGP94 compared to the C3H mammary carcinoma. The higher dose could be explained by the more aggressive nature of the SCCVII tumor model. Supported by The Danish Cancer Society, The Danish Council for Independent Research: Medical Sciences, and OXiGENE Inc. Citation Format: Thomas R. Wittenborn, Michael Stratford, Mary Lynn Trawick, Kevin G. Pinney, David J. Chaplin, Dietmar W. Siemann, Michael R. Horsman. Assessment of anti-tumor activity of the cathepsin L inhibitor, KGP94. [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 1816. doi:10.1158/1538-7445.AM2014-1816

Abstract 321: Autophagy is required for focal adhesion turnover, tumor cell motility and metastasis

Abstract Macro-autophagy is a catabolic process important for degradation of damaged organelles, protein aggregates and intracellular recycling of proteins and lipids. Autophagy is thought to suppress tumor initiation by promoting genome stability and limiting necrosis and inflammation, but conversely is utilized by tumor cells to survive nutrient stress, hypoxia, and cytotoxic therapies, promoting growth of established tumors. However, whether autophagy impacts the metastatic process is not well understood. To examine the role of autophagy in breast cancer metastasis, essential autophagy genes atg5 and atg7 were stably knocked down the aggressive and metastatic 4T1 mouse mammary tumor cell line using shRNA. Inhibition of autophagy did not reduce cell proliferation or survival under stress in vitro or primary tumor growth in vivo, but markedly reduced metastasis to the lungs and liver. Surprisingly, this was not due reduced survival in the circulation or at the secondary site, as atg5 and atg7-deficient cells were able to colonize the lungs to the same extent as control cells when injected into the circulation in a tail vein experimental metastasis assay, suggesting that inhibition of autophagy impairs escape from the primary tumor in this model. Indeed, inhibition of autophagy prevented cell spreading, migration and invasion through collagen in transwell assays of 4T1 cells, MDA-MB-231 human breast cancer cells, and B16.F10 melanoma cells. This was associated with failure of RhoA-mediated protrusion, impaired focal adhesion disassembly, and accumulation of the key focal adhesion component paxillin. We show that paxillin is degraded by autophagy, and siRNA-mediated knockdown of paxillin in autophagy-deficient cells rescued their spreading and motility, suggesting that impaired focal adhesion disassembly and migration in autophagy-deficient cells was the result of failure of autophagic degradation of paxillin. Intriguingly, active RhoA is localized at autophagosomes, and inhibition of RhoA activity inhibits not only cellular protrusion but also autophagic flux in metastatic cells, suggesting that RhoA may be involved in upstream regulation of autophagic degradation of paxillin to promote focal adhesion disassembly. In sum, we have identified a novel and unexpected function for autophagy in focal adhesion disassembly and tumor cell motility that is required for metastasis in vivo. This suggests that while autophagy may also promote the survival of disseminating tumor cells during metastasis, autophagy is in fact required for escape from the primary tumor. Citation Format: Marina N. Sharifi, Christopher Collier, Lauren Drake, Hong Chen, Stephanie Mui, Kay F. Macleod. Autophagy is required for focal adhesion turnover, tumor cell motility and metastasis. [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 321. doi:10.1158/1538-7445.AM2014-321

Inhibition of Established Pancreatic and Triple Negative Breast Tumor Growth by Blocking the Most Downstream Signaling Module, SIAH, in the Oncogenic ERBB/K-RAS Signaling Pathway

Inhibition of Established Pancreatic and Triple Negative Breast Tumor Growth by Blocking the Most Downstream Signaling Module, SIAH, in the Oncogenic ERBB/K-RAS Signaling Pathway

Alpha-galactosylceramide loaded tumor cells in combination with TLR9 agonists induce potent anti-tumor responses in a murine model of colon cancer

Objective To design a new cancer vaccine by using alpha-galactosylceramide （α-Galcer,α-GC） loaded tumor cells in combination with TLR 9 ligand and to evaluate its therapeutic effects on colon canc-er in mice.Methods MC38 cells were transfected with lentivirus （GFP-CD1d） to prepare CD1d-MC38 cells. The expression of CD1d molecules in CD1d-MC38 cells was detected by fluorescence microscopy , RT-PCR and flow cytometry.The sorted CD1d-MC38 cells were loaded with α-Galcer to prepare CD1d-MC38/α-GC complex. Flow cytometry was performed to evaluate the efficiency of combination .A mouse model of colon cancer was es-tablished to investigate the therapeutic effects of α-Galcer loaded tumor cells in combination with TLR 9 ligand （ CD1d-MC38/α-GC＋CpG1826） on colon cancer in mice by analyzing tumor growth and mice survival time .Im-munohistochemical staining was used to detect CD 4＋T and CD8＋T infiltrating lymphocytes in tumor tissues .Re-sults The MC38 cancer cells that expressed CD 1d and GFP were successfully constructed , among which 98.10%±2.53%were positive for CD1d.Moreover, the CD1d-MC38 cells could combine with α-Galcer effec-tively in a dose and time dependent manner .Compared with PBS treated group ,α-GC treated group and TLR9 ligand treated group , the experimental vaccine strategy was sufficient to inhibit the growth of established tumors and prolong survival of tumor-bearing mice （P〈0.01）.Immunohistochemistry analysis revealed that levels of CD4＋T cells and CD8＋T cells in experiment group were significantly higher than those in groups treated with PBS,α-GC and TLR9 ligand （P〈0.01）.Conclusion CD1d-MC38/α-GC in combination with CpG1826 could efficiently inhibit the growth of established tumors and prolong survival of tumor-bearing mice .Immunohisto-chemistry analysis revealed that CD 4＋T cells and CD8＋T cells played important roles in anti-tumor immunity. Key words: iNKT cells; Dendritic cells; α-Galcer; Cancer vaccine

P62/SQSTM1 synergizes with autophagy for tumor growth in vivo.

Autophagy is crucial for cellular homeostasis and plays important roles in tumorigenesis. FIP200 (FAK family-interacting protein of 200 kDa) is an essential autophagy gene required for autophagy induction, functioning in the ULK1-ATG13-FIP200 complex. Our previous studies showed that conditional knockout of FIP200 significantly suppressed mammary tumorigenesis, which was accompanied by accumulation of p62 in tumor cells. However, it is not clear whether FIP200 is also required for maintaining tumor growth and how the increased p62 level affects the growth in autophagy-deficient FIP200-null tumors in vivo. Here, we describe a new system to delete FIP200 in transformed mouse embryonic fibroblasts as well as mammary tumor cells following their transplantation and show that ablation of FIP200 significantly reduced growth of established tumors in vivo. Using similar strategies, we further showed that either p62 knockdown or p62 deficiency in established FIP200-null tumors dramatically impaired tumor growth. The stimulation of tumor growth by p62 accumulation in FIP200-null tumors is associated with the up-regulated activation of the NF-κB pathway by p62. Last, we showed that overexpression of the autophagy master regulator TFEB(S142A) increased the growth of established tumors, which correlated with the increased autophagy of the tumor cells. Together, our studies demonstrate that p62 and autophagy synergize to promote tumor growth, suggesting that inhibition of both pathways could be more effective than targeting either alone for cancer therapy.

Oncogenes and the coagulation system – forces that modulate dormant and aggressive states in cancer

Cancers arise and progress genetically amidst profound perturbations of the microenvironmental and systemic homeostasis. This includes the coagulation system, which is a part of the vascular milieu (niche) that remains under the control of molecular events occurring within the cancer cell genome. Thus, activation of several prototypic oncogenic pathways, such as RAS, EGFR, HER2, MET, SHH and loss of tumor suppressors (PTEN, TP53) alter the expression, activity and vesicular release of coagulation effectors, as exemplified by tissue factor (TF). The cancer-specific determinants of coagulopathy are also illustrated by the emerging link between the expression profiles of coagulation-related genes (coagulome) in glioblastoma multiforme (GBM), medulloblastoma (MB) and possibly other cancers and molecular subtypes of these respective tumors. The state of the coagulome is consequential for growth, metastasis and angiogenesis of established tumors, but could potentially also affect dormant cancer cells. For example, TF expression may trigger awakening of dormant glioma cells in mice in a manner involving recruitment of vascular and inflammatory cells, and resulting in lasting changes in the cancer cell genome and epigenome. Thus, coagulation system effectors could act as both targets and (indirect) inducers of genetic tumor progression, and a better understanding of this link may hold new diagnostic and therapeutic opportunities.

Nod-like receptors: key molecular switches in the conundrum of cancer.

It is believed the immune system can contribute to oncogenic transformation especially in settings of chronic inflammation, be activated during immunosurveillance to destroy early neoplastic cells before they undergo malignant outgrowth, and finally, can assist growth of established tumors by preventing clearance, remodeling surrounding tissue, and promoting metastatic events. These seemingly opposing roles of the immune system at the different stages of cancer development must all be mediated by innate signaling mechanisms that regulate the overall state of immune activation. Recently, the cytosolic nod-like receptor (NLR) pathway of innate immunity has gained a lot of attention in the tumor immunology field due to its known involvement in promoting inflammation and immunity, and conversely, in regulating tissue repair processes. In this review, we present all the current evidence for NLR involvement in the different stages of neoplasia to understand how a single molecular pathway can contribute to conflicting immunological interactions with cancer.

Small GTPase RBJ Mediates Nuclear Entrapment of MEK1/MEK2 in Tumor Progression

Ras-related small GTPases play important roles in cancer. However, the roles of RBJ, a representative of the sixth subfamily of Ras-related small GTPases, in tumorigenesis and tumor progression remain unknown. Here, we report that RBJ is dysregulated in human gastrointestinal cancers and can promote carcinogenesis and tumor progression via nuclear entrapment of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)1/MEK2 and activation of ERK1/ERK2. Nucleus-localized RBJ interacts with MEK/ERK and prolongs the duration of MEK/ERK activation. Rbj deficiency abrogates nuclear accumulation of MEK1/MEK2, attenuates ERK1/ERK2 activation, and impairs AOM/DSS-induced colonic carcinogenesis. Moreover, Rbj knockdown inhibits growth of established tumors. Our data suggest that RBJ may be an oncogenic Ras-related small GTPase mediating nuclear accumulation of active MEK1/MEK2 in tumor progression.

Inhibition of RET Increases the Efficacy of Antiestrogen and Is a Novel Treatment Strategy for Luminal Breast Cancer

Recent findings suggest that combination treatment with antiestrogen and anti-RET may offer a novel treatment strategy in a subset of patients with breast cancer. We investigated the role of RET in potentiating the effects of antiestrogen response and examined whether RET expression predicted the ability for tyrosine kinase inhibitor (TKI) to affect extracellular signal-regulated kinase 1/2 (ERK1/2) activation in primary breast cancer. Growth response, ERK1/2 activation, Ki-67, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were assessed in breast cancer cell lines in vitro and in xenografts with vandetanib and/or tamoxifen. Thirty tumors with matched normal breast tissue were evaluated for RET expression and response to TKI treatment. Vandetanib potentiated the inhibitory effect of tamoxifen in hormone responsive (P = 0.01) and hormone insensitive (P < 0.001) estrogen receptor α (ERα)-positive breast cancer cells. Vandetanib significantly repressed tumorigenesis of MCF-7 xenografts (P < 0.001), which displayed decreased activation of ERK1/2 and AKT. Vandetanib and tamoxifen reduced the growth of established tumors with a greater effect of dual therapy compared with single agent (P = 0.003), with tamoxifen-reducing proliferative index and vandetanib-inducing apoptosis. In primary breast cancers, RET expression correlated with the ERα-positive subtype. Relative decrease in ERK1/2 phosphorylation with TKI treatment was 42% (P < 0.001) in RET-positive tumors versus 14% (P = ns) in RET-negative tumors. Vandetanib potentiated the antigrowth effects of tamoxifen in breast cancer, which was mediated through RET activation. RET predicted response to TKI therapy with minimal effects on ERK1/2 activation in RET-negative tumors. The preclinical data support evaluation of antiestrogen in combination with TKI as a potential treatment strategy for RET-positive luminal breast cancer.

Wild type p53 reactivation: from lab bench to clinic.

The p53 tumor suppressor is the most frequently inactivated gene in cancer. Several mouse models have demonstrated that the reconstitution of the p53 function suppresses the growth of established tumors. These facts, taken together, promote the idea of p53 reactivation as a strategy to combat cancer. This review will focus on recent advances in the development of small molecules which restore the function of wild type p53 by blocking its inhibitors Mdm2 and MdmX or their upstream regulators and discuss the impact of different p53 functions for tumor prevention and tumor eradication. Finally, the recent progress in p53 research will be analyzed concerning the role of p53 cofactors and cellular environment in the biological response upon p53 reactivation and how this can be applied in clinic.