Proteomics discovery of novel cancer serum biomarkers is hindered by the great complexity of serum, patient-to-patient variability, and triggering by the tumor of an acute-phase inflammatory reaction. This host response alters many serum protein levels in cancer patients, but these changes have low specificity as they can be triggered by diverse causes. We addressed these hurdles by utilizing a xenograft mouse model coupled with an in-depth 4-D protein profiling method to identify human proteins in the mouse serum. This strategy ensures identified putative biomarkers are shed by the tumor, and detection of low-abundance proteins shed by the tumor is enhanced because the mouse blood volume is more than a thousand times smaller than that of a human. Using TOV-112D ovarian tumors, more than 200 human proteins were identified in the mouse serum, including novel candidate biomarkers and proteins previously reported to be elevated in either ovarian tumors or the blood of ovarian cancer patients. Subsequent quantitation of selected putative biomarkers in human sera using label-free multiple reaction monitoring (MRM) mass spectrometry (MS) showed that chloride intracellular channel 1, the mature form of cathepsin D, and peroxiredoxin 6 were elevated significantly in sera from ovarian carcinoma patients.
Biomarker discovery; ovarian cancer; cancer biomarkers; xenograft mouse model; proteomics; multidimensional protein profiling
Lymphatic invasion (LI) in primary cutaneous melanomas was recently found to be common. In this study, we evaluated LI as an independent prognostic factor.
This study included 251 patients with vertical growth phase (VGP) primary cutaneous melanomas who had paraffin-fixed lesional tissue and were in a prospective cohort seen between 1972 and 1991, had no clinical evidence of regional nodal disease at diagnosis and had at least ten years of follow-up. Dual immunohistochemistry (IHC) staining was used to detect lymphatic endothelium (podoplanin) and melanoma cells (S-100). Multivariate logistic regression for ten-year metastasis was used to define independent prognostic factors and a prognostic tree was developed to characterize and discriminate risk groups. Kaplan-Meier disese-free survival curves for those with and without LI within current AJCC stages were compared using the log-rank statistic.
LI was observed in 43% (108 of 251) of the study melanomas. The multivariate model for ten-year metastasis identified 4 independent prognostic factors: tumor thickness, mitotic rate (MR), LI, and anatomic site. The prognostic tree identified a group of patients with thin (≤1 mm thick) melanomas and poor prognosis: stage IB melanomas with LI. Survival curves for time to first metastasis demonstrated significantly poorer prognosis for patients with LI compared to those without it for both stages IB and IIA.
LI is common across the range of tumor thicknesses in primary VGP melanomas. It is an independent prognostic factor and significantly increases the risk of metastasis in patients in clinical stages IB and IIA.
Zinc finger E-box binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated senescence, epithelial to mesenchymal transition (EMT) and cancer stem cell function. ZEBs are negatively regulated by members of the miR-200 microRNA family, but precisely how tumor cells expressing ZEBs emerge during invasive growth remains unknown. Here we report that NOTCH3-mediated signaling prevents expansion of a unique subset of ZEB-expressing cells. ZEB expression was associated with the lack of cellular capability of undergoing NOTCH3-mediated squamous differentiation in human esophageal cells. Genetic inhibition of the Notch-mediated transcriptional activity by dominant-negative Mastermind-like1 (DNMAML1) prevented squamous differentiation and induction of Notch target genes including NOTCH3. Moreover, DNMAML1 enriched EMT competent cells exhibited robust upregulation of ZEBs, downregulation of the miR-200 family, and enhanced anchorage independent growth and tumor formation in nude mice. RNA interference (RNAi) experiments suggested the involvement of ZEBs in anchorage independent colony formation, invasion and TGF-β-mediated EMT. Invasive growth and impaired squamous differentiation was recapitulated upon Notch inhibition by DNMAML1 in organotypic 3D culture, a form of human tissue engineering. Together, our findings indicate that NOTCH3 is a key factor limiting the expansion of ZEB-expressing cells, providing novel mechanistic insights into the role of Notch signaling in the cell fate regulation and disease progression of squamous esophageal cancers.
Notch; EMT; squamous cell differentiation; ZEB1; miR-200
Complement activation plays a critical role in controlling inflammatory responses. To assess the role of complement during ovarian cancer progression, we crossed two strains of mice with genetic complement deficiencies with transgenic mice that develop epithelial ovarian cancer (TgMISIIR-TAg). TgMISIIR-TAg mice fully or partially deficient for complement factor 3 (C3) (Tg+C3KO and Tg+C3HET, respectively) or fully deficient for complement factor C5a receptor (C5aR) (Tg+C5aRKO) develop either no ovarian tumors or tumors that were small and poorly vascularized compared to wild-type littermates (Tg+C3WT, Tg+C5aRWT). The percentage of tumor infiltrating immune cells in Tg+C3HET tumors compared to Tg+C3WT controls was either similar (macrophages, B cells, myeloid-derived suppressor cells), elevated (effector T cells), or decreased (regulatory T cells). Regardless of these ratios, cytokine production by immune cells taken from Tg+C3HET tumors was reduced on stimulation compared to Tg+C3WT controls. Interestingly, CD31+ endothelial cell (EC) function in angiogenesis was significantly impaired in both C3KO and C5aRKO mice. Further, using the C5aR antagonist PMX53, tube formation of ECs was shown to be C5a-dependent, possibly through interactions with the VEGF165 but not VEGF121 isoform. Finally, the mouse VEGF164 transcript was underexpressed in C3KO livers compare to C3WT livers. Thus, we conclude that complement inhibition blocks tumor outgrowth by altering EC function and VEGF165 expression.
Because of its restricted distribution in normal tissues and its high expression on tumors of neuroectodermal origin, GD2 ganglioside is an excellent target for active specific immunotherapy. However, GD2 usually elicits low-titered IgM and no IgG or cellular immune responses, limiting its usefulness as a vaccine for cancer patients. We have previously shown that anti-idiotypic monoclonal antibody mimics of GD2 can induce antigen-specific humoral and cellular immunity in mice, but inhibition of tumor growth by the mimics could not be detected.
Methods and results
Here, we isolated two peptides from phage display peptide libraries by panning with GD2-specific mAb ME361. The peptides inhibited binding of the mAb to GD2. When coupled to keyhole limpet hemocyanin (KLH) or presented as multiantigenic peptides in QS21 adjuvant, the peptides induced in mice antibodies binding specifically to GD2 and delayed-type hypersensitive lymphocytes reactive specifically with GD2-positive D142.34 mouse melanoma cells. Induction of delayed-type hypersensitivity (DTH) reaction was dependent on CD4-positive lymphocytes. The immunity elicited by the peptides significantly inhibited growth of GD2-positive melanoma cells in mice.
Our study suggests that immunization with peptides mimicking GD2 ganglioside inhibits tumor growth through antibody and/or CD4-positive T cell-mediated mechanisms. Cytolytic T lymphocytes most likely do not play a role. Our results provide the basis for structural analysis of carbohydrate mimicry by peptides.
GD2; Peptide mimics; Antibodies; DTH; Tumor growth inhibition
We sought to assess the feasibility and reproducibility of performing tissue-based immune characterization of the tumor microenvironment using CT-compatible needle biopsy material. Three independent biopsies were obtained intraoperatively from one metastatic epithelial ovarian cancer lesion of seven consecutive patients undergoing surgical cytoreduction using a 16-gauge core biopsy needle. Core specimens were snap-frozen and subjected to immunohistochemistry (IHC) against human CD3, CD4, CD8 and FoxP3. A portion of the cores was used to isolate RNA for (1) real-time quantitative (q)PCR for CD3, CD4, CD8, FoxP3, IL-10 and TGFβ, (2) multiplexed PCR-based T-cell receptor (TCR) CDR3 V-β region spectratyping and (3) gene expression profiling. Pearson's correlations were examined for immunohistochemistry and PCR gene expression, as well as for gene expression array data obtained from different tumor biopsies. Needle biopsy yielded sufficient tissue for all assays in all patients. IHC was highly reproducible and informative. Significant correlations were seen between the frequency of CD3+, CD8+ and FoxP3+ T cells by IHC with CD3-epsilon, CD8A and FoxP3 gene expression, respectively, by qPCR (r = 0.61, 0.86 and 0.89; all p < 0.05). CDR3 spectratyping was feasible and highly reproducible in each tumor, and indicated a restricted repertoire for specific TCR V-β chains in tumor-infiltrating T cells. Microarray gene expression revealed strong correlation between different biopsies collected from the same tumor. Our results demonstrate a feasible and reproducible method of immune monitoring using CT-compatible needle biopsies from tumor tissue, thereby paving the way for sophisticated translational studies during tumor biological therapy.
ovarian neoplasms; lymphocytes; tumor-infiltrating; adoptive immunotherapy; gene expression profiling; immunohistochemistry
The presence of tumor-infiltrating lymphocytes (TILs) in epithelial ovarian cancer indicates a host antitumor response and is associated with improved survival. We wished to determine the extent to which TIL density differs from site to site within a given patient. We initially studied multiple paired metastases from serous ovarian carcinoma obtained at the time of primary debulking. The expression of genes in specific immune-related pathways was profiled on a pilot set of five patients. We then used immunohistochemistry and quantitative PCR to estimate the density of CD3+, CD8+ and Foxp3+ TILs in these same tumors. To extend the findings to a larger cohort, we semiquantitatively measured intraepithelial and stromal TILs in a tissue microarray (TMA) containing both primary tumors and metastases from 50 patients. In the pilot group, genes related to antimicrobial signaling and TGFβ signaling showed between-site heterogeneity, whereas cytokines and antigen presentation transcripts were more homogeneous in any given patient. IHC and qPCR for T-cell markers were concordant. In the TMA cohort, two-way ANOVA showed that TIL heterogeneity between sites was present in some but not all patients. The stroma of extra-ovarian metastases showed significantly greater TIL infiltration than ovarian sites. A simulation showed that at clinically meaningful levels of precision, up to 3% of patients will be misclassified for intraepithelial TILs by a single biopsy. In conclusion, between-site heterogeneity exists in some patients with metastatic serous ovarian cancer. The predictive value of biopsies should be considered in clinical trial design.
ovarian neoplasms; lymphocytes; tumor-infiltrating; adoptive immunotherapy; metastasis; microarray analysis
Identification and characterization of underlying genetic aberrations could facilitate diagnosis and treatment of ovarian cancer. Copy number analysis using array Comparative Genomic Hybridization (aCGH) on 93 primary ovarian tumors identified PI3K/AKT pathway as the most frequently altered cancer related pathway. Furthermore, survival analyses to correlate gene copy number and mutation data with patient outcome showed that copy number gains of PIK3CA, PIK3CB and PIK3R4 in these tumors were associated with decreased survival. To confirm these findings at the protein level, immunohistochemistry (IHC) for PIK3CA product p110α and p-Akt was performed on tissue microarrays from 522 independent serous ovarian cancers. Overexpression of either of these two proteins was found to be associated with decreased survival. Multivariant analysis from these samples further showed that overexpression of p-AKT and /or p110α is an independent prognostic factor for these tumors. siRNAs targeting altered PI3K/AKT pathway genes inhibited proliferation and induced apoptosis in ovarian cancer cell lines. In addition, the effect of the siRNAs in different cell lines seemed to correlate with the particular genetic alterations that the cell line carries. These results strongly support the utilization of PI3K pathway inhibitors in ovarian cancer. They also suggest identifying the specific component in the PI3K pathway that is genetically altered has the potential to help select the most effective therapy. Both mutation as well as copy number changes can be used as predictive markers for this purpose.
The molecular phenotype of tumor vasculature is different from normal vasculature, offering new opportunities for diagnosis and therapy of cancer, but the identification of tumor-restricted targets remains a challenge. We investigated 13 tumor vascular markers (TVMs) from 50 candidates identified through expression profiling of ovarian cancer vascular cells and selected to be either transmembrane or secreted, and to be either absent or expressed at low levels in normal tissues while overexpressed in tumors, based on analysis of 1,110 normal and tumor tissues from publicly available Affymetrix microarray data. Tumor-specific expression of each TVM was confirmed at the protein level in tumor tissue and/or in serum. Among the 13 TVMs, 11 were expressed on tumor vascular endothelium; the remaining 2 TVMs were expressed by tumor leukocytes. Our results demonstrate that certain transmembrane TVMs such as ADAM12 and CDCP1 are selectively expressed in tumor vasculature and represent promising targets for vascular imaging or anti-vascular therapy of epithelial ovarian cancer, while secreted or shed molecules such as TNFRSF21/DR6 can function as serum biomarkers. We have identified novel tumor-specific vasculature markers which appear promising for cancer serum diagnostics, molecular imaging and/or therapeutic targeting applications and warrant further clinical development.
ovarian; cancer; vascular; biomarkers; diagnostics; serum; expression; profiling
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of squamous cell carcinomas. Common genetic lesions in ESCC include p53 mutations and EGFR overexpression, both of which have been implicated in negative regulation of Notch signaling. In addition, cyclin D1 is overexpressed in ESCC and can be activated via EGFR, Notch and Wnt signaling. To elucidate how these genetic lesions may interact during the development and progression of ESCC, we tested a panel of genetically engineered human esophageal cells (keratinocytes) in organotypic 3D culture (OTC), a form of human tissue engineering. Notch signaling was suppressed in culture and mice by dominant negative Mastermind-like1 (DNMAML1), a genetic pan-Notch inhibitor. DNMAML1 mice were subjected to 4-Nitroquinoline 1-oxide-induced oral-esophageal carcinogenesis. Highly invasive characteristics of primary human ESCC were recapitulated in OTC as well as DNMAML1 mice. In OTC, cyclin D1 overexpression induced squamous hyperplasia. Concurrent EGFR overexpression and mutant p53 resulted in transformation and invasive growth. Interestingly, cell proliferation appeared to be regulated differentially between those committed to squamous-cell differentiation and those invading into the stroma. Invasive cells exhibited Notch-independent activation of cyclin D1 and Wnt signaling. Within the oral-esophageal squamous epithelia, Notch signaling regulated squamous-cell differentiation to maintain epithelial integrity, and thus may act as a tumor suppressor by preventing the development of a tumor-promoting inflammatory microenvironment.
Esophageal squamous cell carcinoma; organotypic 3D culture; EGFR; P53; cyclin D1; Wnt; Notch; squamous-cell differentiation; invasion; 4-Nitroquinoline 1-oxide
Curative eradication of all cells within carcinomas is seldom achievable with chemotherapy alone. This limitation may be partially attributable to tumor cell subpopulations with intrinsic resistance to current drugs. Within squamous cell carcinoma (SCC) cell lines, we previously characterized a subpopulation of mesenchymal-like cells displaying phenotypic plasticity and increased resistance to both cytotoxic and targeted agents. These mesenchymal-like (Ecad-lo) cells are separable from epithelial-like (Ecad-hi) cells based on loss of surface E-cadherin and expression of vimentin. Despite their long-term plasticity, both Ecad-lo and Ecad-hi subsets in short-term culture maintained nearly uniform phenotypes after purification. This stability allowed testing of segregated subpopulations for relative sensitivity to the cytotoxic agent cisplatin in comparison to salinomycin, a compound with reported activity against CD44+CD24− stem-like cells in breast carcinomas. Salinomycin showed comparable efficacy against both Ecad-hi and Ecad-lo cells in contrast to cisplatin, which selectively depleted Ecad-hi cells. An in vivo correlate of these mesenchymal-like Ecad-lo cells was identified by immunohistochemical detection of vimentin-positive malignant subsets across a part of direct tumor xenografts (DTXs) of advanced stage SCC patient samples. Cisplatin treatment of mice with established DTXs caused enrichment of vimentin-positive malignant cells in residual tumors, but salinomycin depleted the same subpopulation. These results demonstrate that mesenchymal-like SCC cells, which resist current chemotherapies, respond to a treatment strategy developed against a stem-like subset in breast carcinoma. Further, they provide evidence of mesenchymal-like subsets being well-represented across advanced stage SCCs, suggesting that intrinsic drug resistance in this subpopulation has high clinical relevance.
EMT; squamous cell carcinoma; head and neck cancer; esophageal cancer; chemotherapy resistance; salinomycin; tumor heterogeneity
The aim of this study was to assess the independent prognostic value of primary tumor mitotic rate compared with other clinical and pathologic features of stages I and II melanoma.
From the American Joint Committee on Cancer (AJCC) melanoma staging database, information was extracted for 13,296 patients with stages I and II disease who had mitotic rate data available.
Survival times declined as mitotic rate increased. Ten-year survival ranged from 93% for patients whose tumors had 0 mitosis/mm2 to 48% for those with ≥ 20/mm2 (P < .001). Mean number of mitoses/mm2 increased as the primary melanomas became thicker (1.0 for melanomas ≤ 1 mm, 3.5 for 1.01 to 2.0 mm, 7.3 for 3.01 to 4.0 mm, and 9.6 for > 8 mm). Ulceration was also associated with a higher mitotic rate; 59% of ulcerated melanomas had ≥ 5 mitoses/mm2 compared with 16% of nonulcerated melanomas (P < .001). In a multivariate analysis of 10,233 patients, the independent predictive factors for survival in order of statistical significance were as follows: tumor thickness (χ2 = 104.9; P < .001), mitotic rate (χ2 = 67.0; P < .001), patient age (χ2 = 48.2; P < .001), ulceration (χ2 = 46.4; P < .001), anatomic site (χ2 = 34.6; P < .001), and patient sex (χ2 = 33.9; P < .001). Clark level of invasion was not an independent predictor of survival (χ2 = 3.2; P = .37).
A high mitotic rate in a primary melanoma is associated with a lower survival probability. Among the independent predictors of melanoma-specific survival, mitotic rate was the strongest prognostic factor after tumor thickness.
The Death Receptor 6 (DR6) protein is elevated in the serum of ovarian cancer patients. We tested DR6 serum protein levels as a diagnostic/predictive biomarker in several epithelial tumors and sarcomas.
DR6 gene expression profiles were screened in publically available arrays of solid tumors. A quantitative immunofluorescent western blot analysis was developed to test the serum of healthy controls and patients with sarcoma, uterine carcinosarcoma, bladder, liver, and pancreatic carcinomas. Change in DR6 serum levels was used to assay the ability of DR6 to predict the response to therapy of sarcoma patients.
DR6 mRNA is highly expressed in all tumor types assayed. Western blot analysis of serum DR6 protein demonstrated high reproducibility (r = 0.97). Compared to healthy donor controls, DR6 serum levels were not elevated in patients with uterine carcinosarcoma, bladder, liver, or pancreatic cancers. Serum DR6 protein levels from adult sarcoma patients were significantly elevated (p<0.001). This was most evident for patients with synovial sarcoma. Change in serum DR6 levels during therapy correlated with clinical benefit from therapy (sensitivity 75%, and positive predictive value 87%).
DR6 may be a clinically useful diagnostic and predictive serum biomarker for some adult sarcoma subtypes.
Diagnosis of sarcoma can be difficult and can lead to improper management of these cancers. DR6 serum protein may be a tool to aid in the diagnosis of some sarcomatous tumors to improve treatment planning. For patients with advanced disease, rising DR6 levels predict non-response to therapy and may expedite therapeutic decision making and reduce reliance on radiologic imaging.
Human squamous cell cancers are the most common epithelially derived malignancies. One example is esophageal squamous cell carcinoma (ESCC), which is associated with a high mortality rate (1) that is related to a propensity for invasion and metastasis (2). Here we report that periostin, a highly expressed cell adhesion molecule, is a key component of a novel tumor invasive signature obtained from an organotypic culture model of engineered ESCC. This tumor invasive signature classifies with human ESCC microarrays, underscoring its utility in human cancer. Genetic modulation of periostin promotes tumor cell migration and invasion as revealed in gain of and loss of function experiments. Inhibition of EGFR signaling and restoration of wild-type p53 function were each found to attenuate periostin, suggesting interdependence of two common genetic alterations with periostin function. Collectively, our studies reveal periostin as an important mediator of ESCC tumor invasion and they indicate that organotypic (3D) culture can offer an important tool to discover novel biologic effectors in cancer.
tumor microenvironment; periostin; EGFR; p53
Regression in the radial growth phase (RGP) of primary cutaneous melanomas is common and has been shown to be an adverse prognostic factor. However, the underlying mechanism is unclear. We performed dual immunohistochemical staining of podoplanin and S100 on paraffin tissues from 321 patients with vertical growth phase (VGP) primary melanomas who had 10 years or more of follow-up. Lymphatic density (LD) and Lymphatic invasion (LI) was quantified and documented. The time to first metastasis and melanoma-specific death from the date of definite treatment was analyzed using univariate and multivariate Cox models. Among the 116 VGP melanomas that had regression in the adjacent RGP, 75 (23%) were classified as complete and 41 (13%) as partial. LD was significantly higher (p<0.001) in the 75 lesions with complete regression (mean ± SD, 23.7 ± 12.3/mm2) compared to the 41 with partial regression (15.5 ± 7.1/mm2) and was lower in 155 areas of adjacent normal dermis (7.3 ± 3.5/mm2) and 69 areas of distant normal dermis (5.5 ± 2.6/mm2). Patients whose lesions had areas of complete regression with LI and either high or low LD or had no LI with high LD, had shorter TFM (HRs = 2.5, 3.8, and 2.5, respectively) and increased risk of melanoma-specific death (HRs=3.1, 1.3 and 3.0, respectively) than those with no LI and low LD or those without areas of complete regression. These data indicate that complete RGP regression is associated with significantly increased LD, and that the adverse prognostic effect of RGP regression is at least partially mediated through lymphangiogenesis and LI in this area.
regression; lymphatic vessel density; lymphatic invasion; melanoma; prognosis
BRAF is an attractive target for melanoma drug development. However, resistance to BRAF inhibitors is a significant clinical challenge. We describe a model of resistance to BRAF inhibitors developed by chronic treatment of BRAFV600E melanoma cells with the BRAF inhibitor SB-590885; these cells are cross resistant to other BRAF-selective inhibitors. Resistance involves flexible switching among the three RAF isoforms, underscoring the ability of melanoma cells to adapt to pharmacological challenges. IGF-1R/PI3K signaling was enhanced in resistant melanomas, and combined treatment with IGF-1R/PI3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells. Increased IGFR-1R and pAKT levels in a post-relapse human tumor sample are consistent with a role for IGF-1R/PI3K-dependent survival in the development of resistance to BRAF inhibitors.
melanoma; BRAF; MEK; IGF-1R; targeted therapy; drug resistance
Melanomas are highly heterogeneous tumors, but the biological significance of their different subpopulations is not clear. Using the H3K4 demethylase JARID1B (KDM5B/PLU-1/RBP2-H1) as a biomarker, we have characterized a small subpopulation of slow-cycling melanoma cells that cycle with doubling times of >4 weeks within the rapidly proliferating main population. Isolated JARID1B-positive melanoma cells give rise to a highly proliferative progeny. Knock-down of JARID1B leads to an initial acceleration of tumor growth followed by exhaustion which suggests that the JARID1B-positive subpopulation is essential for continuous tumor growth. Expression of JARID1B is dynamically regulated and does not follow a hierarchical cancer stem cell model because JARID1B-negative cells can become positive and even single melanoma cells irrespective of selection are tumorigenic. These results suggest a new understanding of melanoma heterogeneity with tumor maintenance as a dynamic process mediated by a temporarily distinct subpopulation.
To revise the staging system for cutaneous melanoma on the basis of data from an expanded American Joint Committee on Cancer (AJCC) Melanoma Staging Database.
The melanoma staging recommendations were made on the basis of a multivariate analysis of 30,946 patients with stages I, II, and III melanoma and 7,972 patients with stage IV melanoma to revise and clarify TNM classifications and stage grouping criteria.
Findings and new definitions include the following: (1) in patients with localized melanoma, tumor thickness, mitotic rate (histologically defined as mitoses/mm2), and ulceration were the most dominant prognostic factors. (2) Mitotic rate replaces level of invasion as a primary criterion for defining T1b melanomas. (3) Among the 3,307 patients with regional metastases, components that defined the N category were the number of metastatic nodes, tumor burden, and ulceration of the primary melanoma. (4) For staging purposes, all patients with microscopic nodal metastases, regardless of extent of tumor burden, are classified as stage III. Micrometastases detected by immunohistochemistry are specifically included. (5) On the basis of a multivariate analysis of patients with distant metastases, the two dominant components in defining the M category continue to be the site of distant metastases (nonvisceral v lung v all other visceral metastatic sites) and an elevated serum lactate dehydrogenase level.
Using an evidence-based approach, revisions to the AJCC melanoma staging system have been made that reflect our improved understanding of this disease. These revisions will be formally incorporated into the seventh edition (2009) of the AJCC Cancer Staging Manual and implemented by early 2010.
Hypoxia-inducible factor 1α (HIF-1α) and HIF-2α display unique and sometimes opposing activities in regulating cellular energy homeostasis, cell fate decisions, and oncogenesis. Macrophages exposed to hypoxia accumulate both HIF-1α and HIF-2α, and overexpression of HIF-2α in tumor-associated macrophages (TAMs) is specifically correlated with high-grade human tumors and poor prognosis. However, the precise role of HIF-2α during macrophage-mediated inflammatory responses remains unclear. To fully characterize cellular hypoxic adaptations, distinct functions of HIF-1α versus HIF-2α must be elucidated. We demonstrate here that mice lacking HIF-2α in myeloid cells (Hif2aΔ/Δ mice) are resistant to lipopolysaccharide-induced endotoxemia and display a marked inability to mount inflammatory responses to cutaneous and peritoneal irritants. Furthermore, HIF-2α directly regulated proinflammatory cytokine/chemokine expression in macrophages activated in vitro. Hif2aΔ/Δ mice displayed reduced TAM infiltration in independent murine hepatocellular and colitis-associated colon carcinoma models, and this was associated with reduced tumor cell proliferation and progression. Notably, HIF-2α modulated macrophage migration by regulating the expression of the cytokine receptor M-CSFR and the chemokine receptor CXCR4, without altering intracellular ATP levels. Collectively, our data identify HIF-2α as an important regulator of innate immunity, suggesting it may be a useful therapeutic target for treating inflammatory disorders and cancer.
We sought to determine whether tumor-infiltrating lymphocytes and/or tumor mitotic activity could identify subgroups of patients with advanced serous epithelial ovarian cancer who would maximally benefit from aggressive surgical cytoreduction.
Snap-frozen specimens from 134 consecutive patients with stage III or IV serous or poorly differentiated ovarian adenocarcinoma undergoing primary debulking surgery from a single US institution were characterized based on CD3+, CD8+, FoxP3+ tumor infiltrating lymphocytes, and Ki67 expression. Kaplan-Meier survival curves were estimated and compared using a log-rank statistic. A multivariate Cox model was used to estimate adjusted hazard ratios (HR). Interactions were modeled using recursive partitioning based on maximal prognostic differentiation.
Brisk intraepithelial CD8+ cells (p=0.035) and low Ki67 expression (p=0.042) portended prolonged survival. T cell infiltration was more likely to occur in tumors with high proliferation index. Patients whose tumors exhibited low Ki67 expression and high intraepithelial CD8+ frequency had a 5-year survival rate of 73.3%. Patients with aggressive tumor behavior, i.e. whose tumors exhibited low frequency of intraepithelial CD8+ T cells or high Ki67 expression were more likely to draw benefit from aggressive surgical cytoreduction. Survival was similar for patients with brisk CD8+ T cells who had optimal or suboptimal debulking. Likewise, survival was similar for patients with low Ki67 expression who had optimal or suboptimal debulking.
These novel interactions of T cells, tumor proliferation index and surgical treatment reveal for the first time that biological prognosticators may be useful for surgical decision making in ovarian cancer.
Ovarian cancer; T cells; surgical cytoreduction; tumor proliferation
Metastasis is a complex multi-step process requiring the concerted action of many genes and is the primary cause of cancer deaths. Pathways that regulate metastasis enhancement and suppression both contribute to tumor dissemination process. In order to identify novel metastasis suppressors, we set up a forward genetic screen in a mouse model. We transduced a genome-wide RNAi library into the non-metastatic 168FARN breast cancer cell line, orthotopically transplanted the cells into mouse mammary fat pads, and then selected for cells that could metastasize to the lung and identified an RNAi for the KLF17 gene. Conversely, we demonstrate that ectopic expression of KLF17 in highly metastatic 4T1 breast cancer cell line inhibited their ability to metastasize from the mammary fat pad to the lung. We also show that suppression of KLF17 expression promotes breast cancer cell invasion and epithelial-mesenchymal transition (EMT) and that KLF17 functions by directly binding to the promoter of Id-1, a key metastasis regulator in breast cancer, to inhibit its transcription. Finally, we demonstrate that KLF17 expression is significantly down-regulated in primary human breast cancer samples and that the combined expression patterns of KLF17 and Id-1 can serve as a potential biomarker for lymph node metastasis in breast cancer.
Cyclophosphamide-based adjuvant chemotherapy is a mainstay of treatment for women with node-positive breast cancer, but is not universally effective in preventing recurrence. Pharmacogenetic variability in drug metabolism is one possible mechanism of treatment failure. We hypothesize that functional single nucleotide polymorphisms (SNPs) in drug metabolizing enzymes (DMEs) that activate (CYPs) or metabolize (GSTs) cyclophosphamide account for some of the observed variability in disease outcomes.
We performed a retrospective cohort study of 350 women enrolled in a multicenter, randomized, adjuvant breast cancer chemotherapy trial (ECOG-2190/INT-0121). Subjects in this trial received standard-dose cyclophosphamide, doxorubicin and fluorouracil (CAF), followed by either observation or high-dose cyclophosphamide and thiotepa with stem cell rescue. We used bone marrow stem cell-derived genomic DNA from archival specimens to genotype CYP2B6, CYP2C9, CYP2D6, CYP3A4, CYP3A5, GSTM1, GSTT1, and GSTP1. Cox regression models were computed to determine associations between genotypes (individually or in combination) and disease-free survival (DFS) or overall survival (OS), adjusting for confounding clinical variables.
In the full multivariable analysis, women with at least one CYP3A4 *1B variant allele had significantly worse DFS than those who were wild-type *1A/*1A (multivariate hazard ratio 2.79; 95% CI 1.52, 5.14). CYP2D6 genotype did not impact this association among patients with estrogen receptor (ER) -positive tumors scheduled to receive tamoxifen.
These data support the hypothesis that genetic variability in cyclophosphamide metabolism independently impacts outcome from adjuvant chemotherapy for breast cancer.
Mammalian sterile 20 (MST1) kinase, a member of the sterile 20 (Ste-20) family of proteins, is a proapoptotic cytosolic kinase that plays an important role in the cellular response to oxidative stress. In this study, we report on the development of a potent and selective MST1 kinase inhibitor based on a ruthenium half-sandwich scaffold. We show that the enantiopure organoruthenium inhibitor, 9E1, has an IC50 value of 45 nM for MST1 and a greater than 25-fold inhibitor selectivity over the related Ste-20 kinases, p21 activated kinase 1 (PAK1), and p21 activated kinase 4 (PAK4) and an almost 10-fold selectivity over the related Thousand and one amino acids kinase 2 (TAO2). Compound 9E1 also displays a promising selectivity profile against unrelated protein kinases, however, the proto-oncogene serine/threonine protein kinase PIM1 (PIM-1) and glycogen synthase kinase 3 (GSK-3β) are inhibited with IC50 values in the low nanomolar range. We also show that 9E1 can inhibit MST1 function in cells. A cocrystal structure of a related compound with PIM-1 and a homology model with MST1 reveals the binding mode of this scaffold to MST1 and provides a starting point for the development of improved MST1 kinase inhibitors for possible therapeutic application.
Liposomal doxorubicin (Doxil) is a cytotoxic chemotherapy drug with a favorable hematologic toxicity profile. Its active drug, doxorubicin, has interesting immunomodulatory properties. Here, the effects of Doxil on surviving tumor cell immunophenotype were investigated.
Using ID8 murine ovarian cancer cells, the immunomodulatory effects of Doxil were studied by measuring its impact on ovarian cancer cell expression of MHC class-I and Fas, and susceptibility to immune attack in vitro. To evaluate the ability of Doxil to cooperate with cancer immunotherapy, the interaction between Doxil and Interleukin 18 (IL-18), a pleiotropic immunostimulatory cytokine, was investigated in vivo in mice bearing ID8-Vegf tumors.
While Doxil killed ID8 tumor cells in a dose-dependent manner, tumor cells escaping Doxil-induced apoptosis upregulated surface expression of MHC-I and Fas, and were sensitized to CTL killing and Fas-mediated death in vitro. We therefore tested the hypothesis that the combination of immunotherapy with Doxil provides positive interactions. Combination IL-18 and Doxil significantly suppressed tumor growth compared with either monotherapy in vivo and uniquely resulted in complete tumor regression and long term antitumor protection in a significant proportion of mice.
These data demonstrate that Doxil favorably changes the immunophenotype of a large fraction of the tumor that escapes direct killing thus creating an opportunity to expand tumor killing by immunotherapy, which can be capitalized through addition of IL-18 in vivo.