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XLSX file - 609K, Supplemental Table 6. Overlap of genes between MMP-3-treated cells and invasive/metastatic pancreatic cancer cells.

PDF file - 95K, Supplemental Table 2. Analysis of Ra.c1b polarity vs MMP3 & Rac1b Staining Intensity

PDF file - 50K

XLSX file - 14K, Supplemental Table 4. Gene Ontology categories enriched in transcriptional profiles of cells treated with MMP-3.

PDF file - 883K, Supplemental Figure 1. MMP3 and Rac1b expression are not correlated with collagen and smooth muscle actin in pancreatic adenocarcinoma cells. Supplemental Figure 2. Association of Rac1b subcellular localization with patient prognosis is not dependent upon patient stage or grade. Supplemental Figure 3. Induction of fibrosis and activation of metaplasia in response to MMP3. Supplemental Figure 4. Meta-analysis of datasets showing overlap of response of PaTu8988T to MMP3 with pancreas biosets. Supplemental Figure 5. Nuclear localization of pSMAD2 in response to MMP3.

XLSX file - 59K, Supplemental Table 3. Gene expression changes in response to MMP-3 treatment.

XLSX file - 364K, Supplemental Table 5. Overlap of genes between MMP-3-treated cells and TGFbeta-treated cells.

PDF file - 110K, Supplemental Table 1. Analysis of Patient Stage vs MMP3 & Rac1b Staining Intensity.

Basal subtype breast cancers have a particularly poor prognosis, with high invasiveness and resistance to most targeted therapies. TGFβ and MYC drive central features of basal breast cancer: TGFβ is an autocrine and paracrine signaling factor that drives cell invasion and metastasis, and MYC is a central regulator of cellular proliferation that is upregulated in many cancer types. We show here that genetic or pharmacologic inhibition of MYC in MCF10A basal breast cells results in increased sensitivity to TGFβ-stimulated invasion and metastasis and also show that this signaling loop is dependent on activation of SRC. Analysis of human breast cancer datasets and additional experiments with breast cancer cell lines further suggest the relevance of this signaling loop in basal, but not luminal, breast cancers. Our results imply precaution should be taken when utilizing therapeutic inhibitors of MYC with basal breast cancer patients as this could lead to increased metastasis; however, simultaneous pharmacologic inhibition of SRC and MYC for these patients could facilitate the antiproliferative effects of MYC inhibition while blocking the consequent promotion of metastasis. Cancer Res; 76(12); 3520–30. ©2016 AACR.

Legends for Supplemental Figures S1-S5.

Responsiveness of different molecular subtypes breast cancer cell lines to TGFβ as measured by PAI-1 expression levels (S1); TGFβ�and MYC knockdown inhibit cell proliferation (S2); MYC reduction potentiates TGFβ induced invasive phenotype in SUM149 cells (S3); Pharmacological inhibition of SRC blocks the effect of TGFβ treatment on increased levels of integrin αvβ3 (S4); Pharmacological inhibition of MYC mimics the effect of MYC knockdown on integrin αvβ3 levels, in a dose dependent manner (S5).

A major clinical challenge of ovarian cancer is the development of malignant ascites accompanied by widespread peritoneal metastasis. In ovarian clear cell carcinoma (OCCC), a challenging subtype of ovarian cancer, this problem is compounded by near-universal primary chemoresistance; patients with advanced stage OCCC thus lack effective therapies and face extremely poor survival rates. Here we show that tumor-cell-expressed serine protease inhibitor Kazal type 1 (SPINK1) is a key driver of OCCC progression and metastasis. Using cell culture models of human OCCC, we find that shRNA silencing of SPINK1 sensitizes tumor cells to anoikis and inhibits proliferation. Knockdown of SPINK1 in OCCC cells also profoundly suppresses peritoneal metastasis in mouse implantation models of human OCCC. We next identify a novel autocrine signaling axis in OCCC cells whereby tumor-cell-produced interleukin-6 (IL-6) regulates SPINK1 expression to stimulate a common protumorigenic gene expression pattern leading to anoikis resistance and proliferation of OCCC cells. We further demonstrate that this signaling pathway can be successfully interrupted with the IL-6Rα inhibitor tocilizumab, sensitizing cells to anoikis in vitro and reducing metastasis in vivo. These results suggest that clinical trials of IL-6 pathway inhibitors in OCCC may be warranted, and that SPINK1 might offer a candidate predictive biomarker in this population.

Ovarian clear cell carcinoma (OCCC) is an understudied poor prognosis subtype of ovarian cancer lacking in effective targeted therapies. Efforts to define molecular drivers of OCCC malignancy may lead to new therapeutic targets and approaches. Among potential targets are secreted proteases, enzymes which in many cancers serve as key drivers of malignant progression. Here, we found that inhibitors of trypsin-like serine proteases suppressed malignant phenotypes of OCCC cell lines. To identify the proteases responsible for malignancy in OCCC, we employed activity-based protein profiling to directly analyze enzyme activity. We developed an activity-based probe featuring an arginine diphenylphosphonate warhead to detect active serine proteases of trypsin-like specificity and a biotin handle to facilitate affinity purification of labeled proteases. Using this probe, we identified active trypsin-like serine proteases within the complex proteomes secreted by OCCC cell lines, including two proteases in common, tissue plasminogen activator and urokinase-type plasminogen activator. Further interrogation of these proteases showed that both were involved in cancer cell invasion and proliferation of OCCC cells and were also detected in in vivo models of OCCC. We conclude the detection of tissue plasminogen activator and urokinase-type plasminogen activator as catalytically active proteases and significant drivers of the malignant phenotype may point to these enzymes as targets for new therapeutic strategies in OCCC. Our activity-based probe and profiling methodology will also serve as a valuable tool for detection of active trypsin-like serine proteases in models of other cancers and other diseases.

Glioblastoma multiforme (GBM) is one of the most aggressive malignant brain tumors and urgently requires the development of new therapeutic strategies. In this study, an innovative hybrid in vitro vascularized GBM-on-a-chip model is presented as a strategic integration of microfluidics and 3D bioprinting technologies. The system can recreate the compartmentalized brain tumor microenvironment, comprising the functional blood brain barrier (BBB) and the adjacent 3D perivascular tumor niche, by selectively mimicking physiological shear stress and cell–cell, cell–matrix mechanical interaction. The GBM-on-a-chip model was evaluated under simulated microgravity (µG) condition as a form of mechanical unloading showing a significant cell morphological and mechanotransduction response thereby indicating that gravitational forces play an important role in glioblastoma mechanical regulation. The proposed GBM-on-a-chip represents a meaningful biological tool for further research in cancer mechanobiology and pre-clinical approach in brain tumor therapy.

Deep space exploration missions need strategies to mitigate the potentially harmful exposure to galactic cosmic radiation. This form of radiation can cause significant damage to biological systems and organisms, which include radiation-induced carcinogenesis in the hematopoietic system. Ongoing studies investigate these effects using cell- and animal-based studies in low earth orbit. The logistic challenges and costs involved with sending biological specimens to space have prompted the development of surrogate ground-based radiation experiments to study the mechanisms of biological injury and cancer risk. However, simulating galactic cosmic radiation has proven difficult and current studies are only partially succeeding at replicating the complexity of this radiation and its downstream injury pathways. Accurate simulation of chronic, low dose galactic radiation will improve our ability to test mitigation strategies such as drug development and improved shielding materials that could be crucial and essential for successful space exploration.

Purpose Sclerosing adenosis (SA), found in ¼ of benign breast disease (BBD) biopsies, is a histological feature characterized by lobulocentric proliferation of acini and stromal fibrosis and confers a two-fold increase in breast cancer risk compared to women in the general population. We evaluated a NanoString-based gene expression assay to model breast cancer risk using RNA derived from formalin-fixed, paraffin-embedded (FFPE) biopsies with SA. Methods The study group consisted of 151 women diagnosed with SA between 1967 and 2001 within the Mayo BBD cohort, of which 37 subsequently developed cancer within 10 years (cases) and 114 did not (controls). RNA was isolated from benign breast biopsies, and NanoString-based methods were used to assess expression levels of 61 genes, including 35 identified by previous array-based profiling experiments and 26 from biological insight. Diagonal linear discriminant analysis of these data was used to predict cancer within 10 years. Predictive performance was assessed with receiver operating characteristic area under the curve (ROC-AUC) values estimated from 5-fold cross-validation. Results Gene expression prediction models achieved cross-validated ROC-AUC estimates ranging from 0.66 to 0.70. Performing univariate associations within each of the five folds consistently identified genes DLK2, EXOC6, KIT, RGS12, and SORBS2 as significant; a model with only these five genes showed cross-validated ROC-AUC of 0.75, which compared favorably to risk prediction using established clinical models (Gail/BCRAT: 0.57; BBD-BC: 0.67). Conclusions Our results demonstrate that biomarkers of breast cancer risk can be detected in benign breast tissue years prior to cancer development in women with SA. These markers can be assessed using assay methods optimized for RNA derived from FFPE biopsy tissues which are commonly available. Electronic supplementary material The online version of this article (doi:10.1007/s10549-017-4441-z) contains supplementary material, which is available to authorized users.

Serine proteases have been implicated as key drivers and facilitators of lung cancer malignancy, and while these proteins represent straightforward targets for therapeutic inhibitors, identification of optimal points for intervention has been complicated by the complex networks in which these enzymes function. Here we implicate a signaling pathway consisting of PRSS3/mesotrypsin and kallikrein-related peptidase 5 (KLK5) in lung adenocarcinoma malignancy. We show that elevated PRSS3/mesotrypsin expression is prognostic for poor outcome for patients with lung adenocarcinoma, and that genetic or pharmacologic targeting of PRSS3/mesotrypsin reduces lung adenocarcinoma cell invasiveness and proliferation. We further show that genetic targeting of KLK5, a known target of PRSS3/mesotrypsin, phenocopies the effect of PRSS3/mesotrypsin knockdown, and also that elevated expression of KLK5 is similarly prognostic for outcome in lung adenocarcinoma. Finally, we use transcriptional profiling experiments to show that PRSS3/mesotrypsin and KLK5 control a common malignancy-promoting pathway. These experiments implicate a potential PRSS3/mesotrypsin-KLK5 signaling module in lung adenocarcinoma and reveal the potential therapeutic benefit of selectively targeting these pathways.

Background Limited effectiveness of therapeutic agents targeting epidermal growth factor receptor (EGFR) in clinical trials using unselected ovarian cancer patients has prompted efforts to more effectively stratify patients who might best benefit from these therapies. A series of studies that have evaluated immunohistochemical (IHC) staining of EGFR in ovarian cancer biopsies has produced unclear results as to the utility of this measure as a prognostic biomarker. Here, we used one of the largest, single institution cohorts to date to determine possible associations of EGFR expression with patient outcome. Methods We performed IHC staining of EGFR in tissue microarrays including nearly 500 patient tumor samples. Staining was classified by subcellular localization (membranous, cytoplasmic) or by automated image analysis algorithms. We also performed a literature review to place these results in the context of previous studies. Results No significant associations were found between EGFR subcellular localization or expression and histology, stage, grade, or outcome. These results were broadly consistent with the consensus of the reviewed literature. Conclusions These results suggest that IHC staining for EGFR may not be a useful prognostic biomarker for ovarian cancer patients. Future studies should pursue other staining methods or analysis in combination with other pathway mediators.

// Christine Mehner 1 , Alexandra Hockla 1 , Erin Miller 1 , Sophia Ran 2 , Derek C. Radisky 1,* and Evette S. Radisky 1,* 1 Department of Cancer Biology, Mayo Clinic, Jacksonville, FL 2 Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL Correspondence: Evette S. Radisky, email: // Derek C. Radisky, email: // Keywords : triple negative breast cancer, basal-like breast cancer, matrix metalloproteinases, invasion, metastasis, mouse orthotopic tumor models, bioluminescence imaging Received : March 3, 2014 Accepted : April 30, 2014 Published : May 1, 2014 Abstract Matrix metalloproteinases (MMPs) have been implicated in diverse roles in breast cancer development and progression. While many of the different MMPs expressed in breast cancer are produced by stromal cells MMP-9 is produced mainly by the tumor cells themselves. To date, the functional role of tumor cell-produced MMP-9 has remained unclear. Here, we show that human breast cancer cell-produced MMP-9 is specifically required for invasion in cell culture and for pulmonary metastasis in a mouse orthotopic model of basal-like breast cancer. We also find that tumor cell-produced MMP-9 promotes tumor vascularization with only modest impact on primary tumor growth, and that silencing of MMP-9 expression in tumor cells leads to an altered transcriptional program consistent with reversion to a less malignant phenotype. MMP-9 is most highly expressed in human basal-like and triple negative tumors, where our data suggest that it contributes to metastatic progression. Our results suggest that MMP9 may offer a target for anti-metastatic therapies for basal-like triple negative breast cancers, a poor prognosis subtype with few available molecularly targeted therapeutic options.

Ovarian cancer remains a challenging disease, with an average 5 year survival rate of 46%. At time of diagnosis, over 70% of patients have advanced metastatic disease, for which there are no effective treatment options. Of patients with metastatic disease, more than 70% of patients develop peritoneal carcinomatosis, making the peritoneum one of the main areas of dissemination. We have previously found that expression of the serine protease inhibitor Kazal type 1 (SPINK1) by ovarian cancer cells drives anoikis resistance, allowing the tumor cells to circumvent apoptosis protocols and facilitating tumor cell metastasis. For this new study we focused on ovarian clear cell carcinoma (OCCC), a histotype of ovarian cancer that has particularly poor prognosis if diagnosed in late stage. Using OCCC cell lines, we found that knockdown of SPINK1 reduces cell survival and stimulates apoptotic pathways in OCCC cells, when cultured under attachment-free conditions to mimic metastatic disease. We then identified Interleukin-6 (IL-6) as an upstream regulator of SPINK1 expression and a potential therapeutic target for metastatic OCCC: we showed that IL-6 silencing reduced SPINK1 mRNA expression and cell survival, while treatment with recombinant IL-6 increased SPINK1 expression and attachment-free survival. We developed a new OCCC mouse model and showed that knockdown of SPINK1 results in significant reduction of metastatic lesions, highlighting the impact of SPINK1 on OCCC metastasis. In ongoing studies we are testing intraperitoneal injections of FDA approved IL-6 inhibitors in a preclinical model to assess impact on abdominal dissemination of tumor cells. By targeting the IL-6 – SPINK1 signaling axis, we aim to develop therapeutic approaches to reduce the metastatic burden and peritoneal carcinomatosis of late stage disease, resulting in better prognosis and survival of patients with ovarian clear cell carcinoma. Citation Format: Christine Mehner, Erin Miller, Mathew A. Coban, Alexandra Hockla, Derek C. Radisky, Evette S. Radisky. Targeting ovarian cancer induced peritoneal carcinomatosis by inhibition of signaling axis between Interleukin-6 (IL-6) and serine protease inhibitor Kazal type 1 (SPINK1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3758.

Basal subtype breast cancers have a particularly poor prognosis, with high invasiveness and resistance to most targeted therapies. TGFβ and MYC drive central features of basal breast cancer: TGFβ is an autocrine and paracrine signaling factor that drives cell invasion and metastasis, and MYC is a central regulator of cellular proliferation that is upregulated in many cancer types. We show here that genetic or pharmacologic inhibition of MYC in MCF10A basal breast cells results in increased sensitivity to TGFβ-stimulated invasion and metastasis and also show that this signaling loop is dependent on activation of SRC. Analysis of human breast cancer datasets and additional experiments with breast cancer cell lines further suggest the relevance of this signaling loop in basal, but not luminal, breast cancers. Our results imply precaution should be taken when utilizing therapeutic inhibitors of MYC with basal breast cancer patients as this could lead to increased metastasis; however, simultaneous pharmacologic inhibition of SRC and MYC for these patients could facilitate the antiproliferative effects of MYC inhibition while blocking the consequent promotion of metastasis. Cancer Res; 76(12); 3520–30. ©2016 AACR.

Patients diagnosed with ovarian cancer face an average 5 year survival rate of 46%; low survival is driven by early and ongoing intraperitoneal dissemination and metastasis of the tumor. Among the different histotypes of ovarian cancer, ovarian clear cell carcinoma (OCCC) has a particularly poor prognosis when diagnosed in late stage, as these tumors tend to be chemoresistant, leaving no effective therapeutic options. Intraperitoneal metastasis requires cells to become anoikis resistant, where apoptosis normally induced by loss of attachment is suppressed. We seek to better understand the mechanisms involved in anoikis resistance in OCCC, which in the future could lead to new therapeutic strategies for these patients. We have found that Serine Protease Inhibitor Kazal type 1 (SPINK1), an endogenous inhibitor of trypsin like serine proteases, is also an important regulator of anoikis resistance in some ovarian cancers. In this study, we aimed to dissect the role of SPINK1 specifically in OCCC, defining its contribution to anoikis resistance using cultured OCCC cell lines and elucidating its mechanism of action. We compared anoikis resistance of cells with endogenous SPINK1 expression versus cells in which SPINK1 was silenced using lentiviral shRNA constructs, when culturing cells on ultra-low attachment plates to mimic cell detachment from the extracellular matrix. We found that knockdown of SPINK1 reduced survival and stimulated apoptotic pathways in OCCC cells grown under detached conditions, implicating SPINK1 in anoikis resistance of OCCC cells. As a secreted protease inhibitor, SPINK1 may be expected to confer anoikis resistance by inhibiting an extracellular serine protease involved in triggering anoikis. Because many proteases represent potential targets of SPINK1, we have designed a strategy using Activity Based Protein Profiling (ABPP) to discover the targets of SPINK1 from among this large pool of candidates. In pilot studies, we have successfully used a novel activity-based probe to covalently label the active sites of serine proteases secreted from OCCC cells. In ongoing efforts, we are optimizing methods to identify labeled proteases with high sensitivity using tandem mass spectrometry. We will then identify those proteases regulated by SPINK1 through comparison of labeled proteomes from OCCC cells with and without SPINK1 treatment,, and candidate proteases will be further analyzed for their role in triggering anoikis. The identification of SPINK1 regulated proteases that are responsible for mediating anoikis in our models will give insight into important mechanisms whereby tumor cells acquire resistance to anoikis in OCCC. In-depth understanding of anoikis resistance, a critical component of ovarian cancer progression and metastasis, may lead to novel biomarkers of disease progression as well as important therapeutic targets. Citation Format: Christine Mehner, Mathew A. Coban, Alexandra Hockla, Derek C. Radisky, Evette S. Radisky. Serine protease inhibitor Kazal type 1 (SPINK1) drives anoikis resistance in ovarian clear cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 442.

Breast, lung, and pancreatic cancers collectively represent one third of all diagnosed tumors and are responsible for almost 40% of overall cancer mortality. Despite improvements in current treatments, efforts to develop more specific therapeutic options are warranted. Here we identify matrix metalloproteinase 3 (MMP3) as a potential target within all three of these tumor types. MMP3 has previously been shown to induce expression of Rac1b, a tumorigenic splice isoform of Rac1. In this study we find that MMP3 and Rac1b proteins are both strongly expressed by the tumor cells of all three tumor types and that expression of MMP3 protein is prognostic of poor survival in pancreatic cancer patients. We also find that MMP3 gene expression can serve as a prognostic marker for patient survival in breast and lung cancer. These results suggest an oncogenic MMP3-Rac1b signaling axis as a driver of tumor progression in three common poor prognosis tumor types, further suggesting that new therapies to target these pathways could have substantial therapeutic benefit.

// Christine Mehner 1 , Ann L. Oberg 2 , Kimberly R. Kalli 3 , Aziza Nassar 4 , Alexandra Hockla 1 , Devon Pendlebury 1 , Magdalena A. Cichon 1 , Krista M. Goergen 2 , Matthew J. Maurer 2 , Ellen L. Goode 5 , Gary L. Keeney 6 , Aminah Jatoi 3 , Miklos Sahin-Toth 7 , John A. Copland 1 , Derek C. Radisky 1 and Evette S. Radisky 1 1 Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, USA 2 Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, MN , USA 3 Department of Medical Oncology, Mayo Clinic, Rochester, MN, USA 4 Department of Laboratory Medicine and Pathology, Mayo Clinic, Jacksonville, FL, USA 5 Division of Epidemiology, Department of Health Sciences Research, Mayo Clinic, Rochester, MN, USA 6 Division of Anatomic Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA 7 Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, USA Correspondence to: Evette S. Radisky, email: // Keywords : SPINK1, ovarian cancer, serine protease inhibitor, EGFR, anoikis Received : July 07, 2015 Accepted : September 14, 2015 Published : September 30, 2015 Abstract Ovarian cancer represents the most lethal tumor type among malignancies of the female reproductive system. Overall survival rates remain low. In this study, we identify the serine protease inhibitor Kazal type 1 (SPINK1) as a potential therapeutic target for a subset of ovarian cancers. We show that SPINK1 drives ovarian cancer cell proliferation through activation of epidermal growth factor receptor (EGFR) signaling, and that SPINK1 promotes resistance to anoikis through a distinct mechanism involving protease inhibition. In analyses of ovarian tumor specimens from a Mayo Clinic cohort of 490 patients, we further find that SPINK1 immunostaining represents an independent prognostic factor for poor survival, with the strongest association in patients with nonserous histological tumor subtypes (endometrioid, clear cell, and mucinous). This study provides novel insight into the fundamental processes underlying ovarian cancer progression, and also suggests new avenues for development of molecularly targeted therapies.

Benign breast disease, a known risk factor for subsequent breast cancer, includes a histological spectrum of lesions that can be subdivided based on the overall impression into non-proliferative lesions, proliferative lesions without atypia, and atypical hyperplasia. Yet little is known about the molecular characteristics of benign breast tissue from African American women, who suffer from disproportionately high breast cancer mortality rates. 72 breast samples from African American women were obtained. RNA was extracted from archival formalin-fixed, paraffin-embedded (FFPE) blocks. Gene expression profiling was performed on these samples using the Affymetrix HTA2.0 exon array chip. A fit linear model using generalized least squares identified probe sets with differential expression in benign-in situ and benign-invasive breast cancer paired lesions. 36 benign and 36 breast cancer paired lesions were included in the study. In the benign-in situ set (n=20), 978 probe sets were differentially expressed (threshold: fold-change, 1.50; p < 0.05). Probe sets mapped to 125 annotated genes, of which 69 (55.2%) were involved in cancer. The benign-invasive set (n=52) identified 351 differentially expressed probe sets that mapped to 84 annotated genes, of which 61 (72.6%) had known cancer significance. Genes involved in cellular growth and proliferation were significantly over-expressed in both benign-in situ and benign-invasive lesions (p-values < 0.0104). In the population of 36 paired samples, differentially expressed genes were found to regulate the estrogen receptor (ESR1) through distinct network associations. Many genes are differentially expressed by benign and breast cancer lesions, and in situ/invasive histology contribute to the distinct molecular characteristics and pathways of breast malignancy. Our findings highlight the first study to assess expression profiling of paired benign and breast cancer lesions among African American women. Citation Format: Andreana Natalie Holowatyj, Julie J. Ruterbusch, Rouba Ali-Fehmi, Sudeshna Bandyopadhyay, Christine Mehner, Melody Stallings Mann, Gregory Dyson, Derek Radisky, Michele L. Cote. Expression profiling of paired benign and breast cancer lesions in African American women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4253. doi:10.1158/1538-7445.AM2017-4253

Pancreatic ductal adenocarcinoma (PDA) arises at the convergence of genetic alterations in KRAS with a fostering microenvironment shaped by immune cell influx and fibrotic changes; identification of the earliest tumorigenic molecular mediators evokes the proverbial chicken and egg problem. Matrix metalloproteinases (MMP) are key drivers of tumor progression that originate primarily from stromal cells activated by the developing tumor. Here, MMP3, known to be expressed in PDA, was found to be associated with expression of Rac1b, a tumorigenic splice isoform of Rac1, in all stages of pancreatic cancer. Using a large cohort of human PDA tissue biopsies specimens, both MMP3 and Rac1b are expressed in PDA cells, that the expression levels of the two markers are highly correlated, and that the subcellular distribution of Rac1b in PDA is significantly associated with patient outcome. Using transgenic mouse models, coexpression of MMP3 with activated KRAS in pancreatic acinar cells stimulates metaplasia and immune cell infiltration, priming the stromal microenvironment for early tumor development. Finally, exposure of cultured pancreatic cancer cells to recombinant MMP3 stimulates expression of Rac1b, increases cellular invasiveness, and activation of tumorigenic transcriptional profiles. Implications: MMP3 acts as a coconspirator of oncogenic KRAS in pancreatic cancer tumorigenesis and progression, both through Rac1b-mediated phenotypic control of pancreatic cancer cells themselves, and by giving rise to the tumorigenic microenvironment; these findings also point to inhibition of this pathway as a potential therapeutic strategy for pancreatic cancer. Mol Cancer Res; 12(10); 1430–9. ©2014 AACR.

Cancers become significantly more dangerous when the tumor progresses from in situ, or contained, to an invasive state, in which the cancer cells acquire the ability to pass through the surrounding basement membrane (BM), a specialized extracellular matrix (ECM) that provides structure and contextual information to the underlying tissue. While the majority of tumors are carcinomas, derived from epithelial cells, it is the stromal cells surrounding the epithelial-derived tumor cells, including fibroblasts and myofibroblasts, vasculature, and immune cells, that are largely responsible for the production and remodeling of the ECM. Here, we will discuss myofibroblasts as key effectors of tumor progression, focusing on recent advances in breast and pancreatic carcinoma, showing how myofibroblasts may function properly in normal tissue remodeling and wound-healing processes, how in the tumor context they can drive cancer invasion and metastasis, and how the pathogenic functions of myofibroblasts may be targeted therapeutically.

Excess proteolytic activity of matrix metalloproteinases (MMPs) contributes to the development of arthritis, cardiovascular diseases and cancer progression, implicating these enzymes as therapeutic targets. While many small molecule inhibitors of MMPs have been developed, clinical uses have been limited, in part by toxicity and off-target effects. Development of the endogenous tissue inhibitors of metalloproteinases (TIMPs) as recombinant biopharmaceuticals represents an alternative therapeutic approach; however, the short plasma half-life of recombinant TIMPs has restricted their potential in this arena. To overcome this limitation, we have modified recombinant human TIMP-1 (rhTIMP-1) by PEGylation on lysine residues. We analyzed a mixture of mono- and di-PEGylated rhTIMP-1 species modified by attachment of 20 kDa mPEG chains (PEG(20K)-TIMP-1), as confirmed by SELDI-TOF mass spectrometry. This preparation retained complete inhibitory activity toward the MMP-3 catalytic domain and partial inhibitory activity toward full length MMP-9. Pharmacokinetic evaluation showed that PEGylation extended the plasma half-life of rhTIMP-1 in mice from 1.1 h to 28 h. In biological assays, PEG(20K)-TIMP-1 inhibited both MMP-dependent cancer cell invasion and tumor cell associated gelatinase activity. Overall these results suggest that PEGylated TIMP-1 exhibits improved potential for development as an anti-cancer recombinant protein therapeutic, and additionally may offer potential for clinical applications in the treatment of other diseases.

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