Immune-regulated pathways influence multiple aspects of cancer development. In this article we demonstrate that both macrophage abundance and T-cell abundance in breast cancer represent prognostic indicators for recurrence-free and overall survival. We provide evidence that response to chemotherapy is in part regulated by these leukocytes; cytotoxic therapies induce mammary epithelial cells to produce monocyte/macrophage recruitment factors, including colony stimulating factor 1 (CSF1) and interleukin-34, which together enhance CSF1 receptor (CSF1R)–dependent macrophage infiltration. Blockade of macrophage recruitment with CSF1R-signaling antagonists, in combination with paclitaxel, improved survival of mammary tumor–bearing mice by slowing primary tumor development and reducing pulmonary metastasis. These improved aspects of mammary carcinogenesis were accompanied by decreased vessel density and appearance of antitumor immune programs fostering tumor suppression in a CD8⁺ T-cell–dependent manner. These data provide a rationale for targeting macrophage recruitment/response pathways, notably CSF1R, in combination with cytotoxic therapy, and identification of a breast cancer population likely to benefit from this novel therapeutic approach. Significance: These findings reveal that response to chemotherapy is in part regulated by the tumor immune microenvironment and that common cytotoxic drugs induce neoplastic cells to produce monocyte/macrophage recruitment factors, which in turn enhance macrophage infiltration into mammary adenocarcinomas. Blockade of pathways mediating macrophage recruitment, in combination with chemotherapy, significantly decreases primary tumor progression, reduces metastasis, and improves survival by CD8⁺ T-cell–dependent mechanisms, thus indicating that the immune microenvironment of tumors can be reprogrammed to instead foster antitumor immunity and improve response to cytotoxic therapy. Cancer Discovery; 1(1); 54–67. ©2011 AACR. This article is highlighted in the In This Issue feature, p. 4

MicroRNAs (miRNAs) are small, noncoding RNAs that can contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies have also linked different sets of miRNAs to metastasis through either the promotion or suppression of this malignant process. Interestingly, epigenetic silencing of miRNAs with tumor suppressor features by CpG island hypermethylation is also emerging as a common hallmark of human tumors. Thus, we wondered whether there was a miRNA hypermethylation profile characteristic of human metastasis. We used a pharmacological and genomic approach to reveal this aberrant epigenetic silencing program by treating lymph node metastatic cancer cells with a DNA demethylating agent followed by hybridization to an expression microarray. Among the miRNAs that were reactivated upon drug treatment, miR-148a, miR-34b/c, and miR-9 were found to undergo specific hypermethylation-associated silencing in cancer cells compared with normal tissues. The reintroduction of miR-148a and miR-34b/c in cancer cells with epigenetic inactivation inhibited their motility, reduced tumor growth, and inhibited metastasis formation in xenograft models, with an associated down-regulation of the miRNA oncogenic target genes, such as C-MYC, E2F3, CDK6, and TGIF2. Most important, the involvement of miR-148a, miR-34b/c, and miR-9 hypermethylation in metastasis formation was also suggested in human primary malignancies ( n = 207) because it was significantly associated with the appearance of lymph node metastasis. Our findings indicate that DNA methylation-associated silencing of tumor suppressor miRNAs contributes to the development of human cancer metastasis.

Tumor-infiltrating immune cells can promote chemoresistance and metastatic spread in aggressive tumors. Consequently, the type and quality of immune responses present in the neoplastic stroma are highly predictive of patient outcome in several cancer types. In addition to host immune responses, intrinsic tumor cell activities that mimic stem cell properties have been linked to chemoresistance, metastatic dissemination, and the induction of immune suppression. Cancer stem cells are far from a static cell population; rather, their presence seems to be controlled by highly dynamic processes that are dependent on cues from the tumor stroma. However, the impact immune responses have on tumor stem cell differentiation or expansion is not well understood. In this study, we show that targeting tumor-infiltrating macrophages (TAM) and inflammatory monocytes by inhibiting either the myeloid cell receptors colony-stimulating factor-1 receptor (CSF1R) or chemokine (C–C motif) receptor 2 (CCR2) decreases the number of tumor-initiating cells (TIC) in pancreatic tumors. Targeting CCR2 or CSF1R improves chemotherapeutic efficacy, inhibits metastasis, and increases antitumor T-cell responses. Tumor-educated macrophages also directly enhanced the tumor-initiating capacity of pancreatic tumor cells by activating the transcription factor STAT3, thereby facilitating macrophage-mediated suppression of CD8⁺ T lymphocytes. Together, our findings show how targeting TAMs can effectively overcome therapeutic resistance mediated by TICs. Cancer Res; 73(3); 1128–41. ©2012 AACR.

Weighted gene coexpression network analysis (WGCNA) is a powerful ‘guilt-by-association’-based method to extract coexpressed groups of genes from large heterogeneous messenger RNA expression data sets. We have utilized WGCNA to identify 11 coregulated gene clusters across 2342 breast cancer samples from 13 microarray-based gene expression studies. A number of these transcriptional modules were found to be correlated to clinicopathological variables (e.g. tumor grade), survival endpoints for breast cancer as a whole (disease-free survival, distant disease-free survival and overall survival) and also its molecular subtypes (luminal A, luminal B, HER2+ and basal-like). Examples of findings arising from this work include the identification of a cluster of proliferation-related genes that when upregulated correlated to increased tumor grade and were associated with poor survival in general. The prognostic potential of novel genes, for example, ubiquitin-conjugating enzyme E2S (UBE2S) within this group was confirmed in an independent data set. In addition, gene clusters were also associated with survival for breast cancer molecular subtypes including a cluster of genes that was found to correlate with prognosis exclusively for basal-like breast cancer. The upregulation of several single genes within this coexpression cluster, for example, the potassium channel, subfamily K, member 5 (KCNK5) was associated with poor outcome for the basal-like molecular subtype. We have developed an online database to allow user-friendly access to the coexpression patterns and the survival analysis outputs uncovered in this study (available at http://glados.ucd.ie/Coexpression/ ).