Citations

Epithelial–mesenchymal transition (EMT) is a form of epithelial plasticity implicated in fibrosis and tumor metastasis. Here we show that the mechanical rigidity of the microenvironment plays a pivotal role in the promotion of EMT by controlling the subcellular localization and downstream signaling of Rac GTPases. Soft substrata, with compliances comparable to that of normal mammary tissue, are protective against EMT, whereas stiffer substrata, with compliances characteristic of breast tumors, promote EMT. Rac1b, a highly activated splice variant of Rac1 found in tumors, localizes to the plasma membrane in cells cultured on stiff substrata or in collagen-rich regions of human breast tumors. At the membrane, Rac1b forms a complex with NADPH oxidase and promotes the production of reactive oxygen species, expression of Snail, and activation of the EMT program. In contrast, soft microenvironments inhibit the membrane localization of Rac1b and subsequent redox changes. These results reveal a novel mechanotransduction pathway in the regulation of epithelial plasticity via EMT.

Abstract Matrix metalloproteinase 10 (Mmp10; stromelysin 2) is a member of a large family of structurally related matrix metalloproteinases, many members of which have been implicated in tumor progression, invasion and metastasis. However, emerging evidence suggests a possible role for Mmps in tumor initiation. Here we reveal an unexpected role for Mmp10 in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). Mouse lung cancer cell cultures enriched in stem-like cells grow as undifferentiated tumor “oncospheres” that express elevated levels of the cancer stem cell markers Aldh1, CD133, Nanog, Notch3, Notch 4, Hey 1 and Hey 2. These cells also express elevated levels of Mmp10 mRNA and secrete elevated levels of Mmp10 protein. Functionally, these lung CSCs exhibit self-renewal, the ability to clonally expand, enhanced transforming potential in vitro, and enhanced tumorigenic properties when injected orthotopically into the lungs of syngeneic mice. RNAi-mediated knockdown of Mmp10 in these cells leads to a loss of stem cell marker gene expression and a dramatic inhibition of oncosphere growth, clonal expansion, transformed growth in vitro, and lung tumor formation and metastasis in vivo. In contrast, oncospheres implanted into syngeneic non-transgenic or Mmp10−/− mice show no significant difference in tumor initiation, growth or metastasis, demonstrating the importance of Mmp10 produced by cancer cells rather than the tumor microenvironment in lung tumor initiation and maintenance. Analysis of gene expression data from human tumors demonstrates that Mmp10 is elevated in many human tumor types including lung, head and neck, esophageal, colon, breast, melanoma, bladder, cervical, ovarian, prostate and brain. Furthermore, gene set enhancement analysis (GSEA) demonstrates that elevated Mmp10 expression correlates with tumor metastasis and with cancer stem-like genomic signatures in human lung tumors. Thus, Mmp10 is required for maintenance of a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in lung cancer. Our data demonstrate for the first time that Mmp10 is a critical lung cancer stem cell gene and novel therapeutic target for lung cancer stem cells. 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 LB-204. doi:1538-7445.AM2012-LB-204

Abstract SHP2 is a protein tyrosine phosphatase encoded by the PTPN11 gene. SHP2 is normally auto-inhibited by its regulatory N-SH2 domain. It is activated by several receptor tyrosine kinases and positively regulates the RAS/ERK and other signaling pathways by mechanisms that remain incompletely defined. Furthermore, gain-of-function activating SHP2 mutants have been found in human hematologic malignancies and solid tumors, including lung, colon, and prostate cancer. Previous studies have demonstrated the oncogenic activity of gain-of-function SHP2 mutants in the hematopoietic system. To investigate the biological functions of mutant SHP2 in vivo, we generated transgenic mice harboring a tetO-SHP2E76K transgene for tetracycline-inducible expression of the constitutively active SHP2E76K mutant. This allows us to examine the roles of the cancer-associated, Glu-76 mutant SHP2 in various tissues in bitransgenic mice. tetO-SHP2E76K mice were crossed with CCSP-rtTA or SPC-rtTA mice for inducible expression of SHP2E76K in the lung type II pneumocytes. Elevated levels of active Erk1/2 and Src were detected in the lungs of bitransgenic mice induced with doxycycline. Focal hyperplasia of bronchioloalveolar epithelial cells was observed within one month of doxycycline induction in bitransgenic mice, which progressed to more extensive atypical adenomous hyperplasia and adenoma after six months. These results suggest that Shp2 is a proto-oncogene of lung carcinoma. 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 4005. doi:1538-7445.AM2012-4005

Rac1b mediates the matrix metalloproteinase–induced epithelial-mesenchymal transition in lung adenocarcinoma and is a potential therapeutic target in early-stage lung cancer.

Abstract Breast cancer recurrence is believed to be caused by a potently tumorigenic sub-population of cancer cells possessing stem cell attributes of resistance to chemotherapy, radiation and other forms of treatment. Recently, we and others have reported the generation of breast cancer stem cell (BCSC) characteristics by inducers of epithelial to mesenchymal transition (EMT). Both EMT and acquisition of BCSC properties are therefore important steps in cancer progression, although the physiological process by which BCSCs arise in vivo remains unclear. We show here that exposure to TGFβ and TNFα induces a stable BCSC phenotype, characterized by self-renewal, greatly increased tumorigenicity, and increased resistance to oxaliplatin, etoposide and paclitaxel. We further found that the TGFβ/TNFα-derived cells showed downregulated expression of Claudins 3, 4 and 7 and the luminal marker KRT18, demonstrating a shift to the claudin low molecular subtype, a recently identified breast cancer subtype characterized by the expression of mesenchymal and stem cell-associated markers and correlation with poor prognosis. Furthermore, pathway analyses of the generated BCSCs showed deregulation of a number of breast cancer-associated genes and pathways including inflammatory cytokine and receptors, chemokines, homeobox, hedgehog, notch and metastases genes. These results confirm the role of EMT and BCSCs in resistance of breast cancer to conventional chemotherapy and identify novel target genes and pathways for validation as potential therapeutic targets. Our demonstration that stable BCSCs can be produced by specific microenvironmental signals also provides a valuable tool for studying cancer stem cells, as well as a means for screening potential cancer drugs for efficacy of targeting cancer stem cells to prevent recurrence. 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 3341. doi:10.1158/1538-7445.AM2011-3341

Abstract Epimorphin is a stromally produced factor that plays a role in normal mammary branching morphogenesis, but deregulated expression of epimorphin can have oncogenic consequences, although the oncogenic processes activated by epimorphin have not been elucidated. We had previously found that treatment of cultured mammary epithelial cells with epimorphin blocked activation of cell death pathways by a variety of apoptotic stimuli. These results implicated a potential role for epimorphin for inhibition of cellular apoptosis, a critical stage of tumor development. To identify potential mediators by which epimorphin could be affecting cell death pathways, we used a transcriptional profiling approach comparing mammary epithelial cells treated with epimorphin versus untreated cells. We found that epimorphin treatment led to significantly increased expression of IKBKE, which inhibits apoptosis through activation of NF-kappaB and which has recently been characterized as a key breast cancer oncogene. We found that epimorphin specifically blocks induction of apoptosis and activation of caspase-3 in cells treated with tumor necrosis factor-alpha, and that this activity was linked to epimorphin-mediated activation of IKBKE. We also found that knockdown of IKBKE blocked epimorphin-mediated induction of branching morphogenesis, implicating activation of IKBKE by epimorphin as a critical function for its normal morphogenic activities as well. These studies provide insight into an additional and unexpected linkage between mammary gland developmental processes and breast cancer progression. 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 4774. doi:10.1158/1538-7445.AM2011-4774

Abstract Most breast cancer deaths are the consequence of metastasis from the primary site. In order to metastasize, tumor cells must escape the physical barriers that contain the growing primary tumor, enter the vascular system, exit the vascular system at a distant point, and colonize a distant organ. An important mediator of metastasis is matrix metalloproteinase-9, a member of the MMP family that has been found to be upregulated both in breast cancer cells and in the surrounding tumor stroma. Studies using animal models have sometimes produced apparently contradictory findings, due in part to methodology in which MMP-9 expression is suppressed throughout the host organism and throughout the course of tumor initiation and progression. To define the specific role of tumor cell-produced MMP-9 in metastasis of human breast cancer, we implemented a bioluminescent imaging animal model of metastasis in which luciferase-expressing MDA-MB-231 cells, infected either with control nontarget lentivirus (MDA-MB-231-NT) or with shRNA lentivirus targeting MMP-9 (MDA-MB-231-MMP9-KD), were orthotopically implanted into the mammary fat pads of immunocompromised mice. Tumor growth and development of lung metastases were assessed over 10 weeks using an IVIS imager. At 11 weeks mice were sacrificed, and tumor progression was assessed by ex vivo lung imaging and by immunohistochemical processing of the tumors and the lungs. Imaging studies revealed that primary tumors developing from MMP-9 knockdown cells grew more slowly throughout the course of the study (ranging from 25% to 50%), and that while the majority of the control group developed lung metastases, none of the mice injected with MDA-MB-231-MMP9-KD cells showed metastatic progression. After sacrifice, the measured tumor weight showed a median of 99 mg for the MDA-MB-231-MMP9-KD and 158 mg for the MDA-MB-231-NT tumors, showing that knockdown of MMP-9 inhibited tumor growth. Ex vivo imaging of the lungs confirmed the in vivo studies: no metastases were detected from MDA-MB-231-MMP9-KD cells, while all lungs of the MDA-MB-231-NT injected mice had detectable metastases (p=0.0079). Similarly, a single level immunohistochemical correlate found no metastases in the MDA-MB-231-MMP9-KD mice, while 3 of 4 mice with the non-target control cells showed large metastases (median 7×103 μm2). Our results demonstrate that expression of MMP-9 by human breast cancer cells plays a significant role in the progression of tumor growth and metastasis and provide insight into the potential of MMP-9 as a target for therapeutic intervention. 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 1497. doi:10.1158/1538-7445.AM2011-1497

Abstract Fusion genes and fusion gene products are widely employed as biomarkers and therapeutic targets in hematopoietic cancers, but their applications have yet to be appreciated in solid tumors. Here, we report the use of SnowShoes-FTD, a powerful new analytic pipeline that can identify fusion transcripts and assess their redundancy and tumor subtype-specific distribution in primary tumors. In a study of primary breast tumors, SnowShoes-FTD was used to analyze paired-end mRNA-Seq data from a panel of estrogen receptor (ER)+, HER2+, and triple-negative primary breast tumors, identifying tumor-specific fusion transcripts by comparison with mRNA-Seq data from nontransformed human mammary epithelial cell cultures plus the Illumina Body Map data from normal tissues. We found that every primary breast tumor that was analyzed expressed one or more fusion transcripts. Of the 131 tumor-specific fusion transcripts identified, 86 were “private” (restricted to a single tumor) and 45 were “redundant” (distributed among multiple tumors). Among the redundant fusion transcripts, 7 were unique to ER+ tumors and 8 were unique to triple-negative tumors. In contrast, none of the redundant fusion transcripts were unique to HER2+ tumors. Both private and redundant fusion transcripts were widely expressed in primary breast tumors, with many mapping to genomic loci implicated in breast carcinogenesis and/or risk. Our finding that some fusion transcripts are tumor subtype-specific suggests that these entities may be critical determinants in the etiology of breast cancer subtypes, useful as biomarkers for tumor stratification, or exploitable as cancer-specific therapeutic targets. Cancer Res; 72(8); 1921–8. ©2012 AACR.

Abstract p16, a nuclear protein encoded by the p16INK4a gene, is a regulator of cell-cycle regulation. Previous studies have shown that expression of p16 in tissue biopsies of patients with ductal carcinoma in situ (DCIS) is associated with increased risk of breast cancer, particularly when considered in combination with other markers such as Ki-67 and COX-2. Here, we evaluated how expression of p16 in breast tissue biopsies of women with atypical hyperplasia (AH), a putative precursor lesion to DCIS, is associated with subsequent development of cancer. p16 expression was assessed by immunohistochemistry in archival sections from 233 women with AH diagnosed at the Mayo Clinic. p16 expression in the atypical lesions was scored by percentage of positive cells and intensity of staining. We also studied coexpression of p16, with Ki-67 and COX-2, biomarkers of progression in AH. Risk factor and follow-up data were obtained via study questionnaire and medical records. Forty-seven patients (20%) developed breast cancer with a median follow-up of 14.5 years. Staining of p16 was increased in older patients relative to younger patients (P = 0.0025). Although risk of developing breast cancer was not associated with increased p16 expression, joint overexpression of Ki-67 and COX-2 was found to convey stronger risk of breast cancer in the first 10 years after diagnosis as compared with one negative marker (P < 0.01). However, the addition of p16 levels did not strengthen this association. p16 overexpression, either alone or in combination with COX-2 and Ki-67, does not significantly stratify breast cancer risk in women with AH. Cancer Prev Res; 4(12); 1953–60. ©2011 AACR.