New player in tumor‐stromal interaction: Granulin as a novel therapeutic target for pancreatic ductal adenocarcinoma liver metastasis
Authors: Misako Sato‐Matsubara, Norifumi Kawada
Journal: Hepatology (2016)

Nielsen et al.1 recently described a new mechanism for tumor-stromal interaction in the metastatic progression of pancreatic ductal adenocarcinoma (PDAC) in the liver through granulin that is secreted by metastasis-associated macrophages (MAMs).1 MAM-derived granulin transactivates hepatic stellate cells (hStCs), which leads to the production of periostin and extracellular matrix (ECM) at the metastatic site. The present report emphasizes the importance of the interaction between MAMs and hStCs-derived myofibroblasts (MFs) through granulin and periostin in the development of the metastatic microenvironment and suggests that these factors are potential drug targets against PDAC liver metastasis. PDAC is one of the most aggressive cancers and exhibits a very poor prognosis. More than 80% of patients have inoperable metastatic tumor at the time of diagnosis, and the cancer frequently recurs within 1 year of extirpative surgery of the primary site. The 5-year survival rate of PDAC remains approximately 6%, with most patients dying because of metastasis to distant organs.2 In general, PDAC spreads rapidly to nearby lymph nodes, and most of the disseminated cancer cells reach the liver and eventually spread further. Regardless of the tumor type, cancer cells recruit immune cells and fibroblasts by producing various growth factors and cytokines, such as colony-stimulating factor 1 (CSF1), transforming growth factor beta 1 (TGF-β1), C-C motif chemokine ligand (CCL) 2, CCL7, and interleukin (IL)-6. These factors stimulate circulating bone marrow (BM)-derived monocytes and resident fibroblasts (and in the case of the liver, hSteCs) to differentiate into macrophages and alpha-smooth muscle actin (αSMA)+-MFs, respectively, thereby establishing a metastatic microenvironment. Because tumors exhibit diverse microenvironments, the therapeutic targeting of tumor-associated stromal cells, which are genetically stable, has both beneficial and adverse consequences for tumorigenesis.3 Therefore, it is important to understand the nature of their stromal constitution in each tumor. Nielsen et al.1 clarified the role of MAMs and hStC-derived MFs as critical contributors at secondary metastatic sites of PDAC (Fig. 1). Nielsen et al. demonstrated that stromal cells accumulated at the metastatic site in human liver biopsies of PDAC patients compared to healthy volunteers. CD45+-immune cells, CD68+-macrophages, αSMA+/platelet-derived growth factor receptor+-MFs, and connective tissues were detected as components of the PDAC metastatic environment. The immune cells were accumulating at the metastasis site in a mouse model generated by intrasplenic injection of PDAC cells isolated from KrasG12D; Trp53R172H; Pdx1-Cre (KPC) mice. The metastasis involved CD11b+F4/80negLy6C+LY6GnegCCR2+ inflammatory monocytes (IMs), CD11b+F4/80+Ly6CnegCCR2+ MAMs, and αSMA+-MFs. MAMs originate from BM-derived hematopoietic cells and not from resident Kupffer cells (KCs), whereas αSMA+-MFs originated from resident hStCs. The depletion of MAMs in vivo using clodronate liposomes or genetic deficiency of PI3Ky, which is mainly expressed in myeloid cells and necessary for their recruitment to the tumor site,4 induced a marked reduction in the number of αSMA+-MFs and impaired the growth of disseminated cancer cells. Thus, MAMs play essential roles in MF activation and PDAC metastatic growth. To identify the MAM-derived factor that regulates MF activation, human THP-1-derived macrophages were exposed to tumor conditioned medium (CM) from the pancreatic cancer cell line, Panc1, and then used for secretome analysis, which led to the identification of granulin as a highly expressed glycoprotein. Granulin is secreted from BM-derived MAMs, but not from resident KCs and trans-differentiates hStCs to αSMA+-MFs. PDAC metastatic growth was significantly diminished in granulin-null mice compared to wild-type mice, and the abolishment of αSMA+-MF accumulation was observed without a reduction in the total number of MAMs. Next, Nielsen et al. performed another secretome analysis for human fibroblasts preincubated with macrophage CM and confirmed the induction of ECM component periostin. Notably, periostin production by hStCs was strictly regulated by granulin, and periostin expression was markedly up-regulated in spontaneous hepatic metastatic lesions in both human and murine tissues compared to healthy controls. Moreover, the investigators reported elevated levels of granulin in circulating IMs (CD14hiCD16neg) from metastatic PDAC patients and KPC mice, suggesting that granulin is a possible predictive biomarker of PDAC metastasis. These new findings on the role of MAMs and hStC-derived MFs in the metastatic outgrowth of PDAC raise several interesting questions. First, which factor initiates the attraction of BM-derived macrophages to the PDAC metastatic site in the liver? In addition to tumor-derived CSF1, TGF-β1, CCL2, CCL7, and IL-6, recent studies have found that different classes of systemic mediators, including exosomes and ECM-remodeling enzymes such as matrix metalloproteinases, and heparanase, are involved in the recruitment of BM-derived cells to the secondary metastatic sites of infiltrating cancer cells.3 Second, how does PDAC interact with MAMs to induce granulin expression? Granulins form a family of glycosylated peptides that are cleaved from a single 88-kDa precursor protein, progranulin, into smaller 6-kDa peptides, known as granulin A, granulin B, granulin C, etc., which either inhibit or stimulate cell growth in normal development, wound healing, and tumorigenesis.5 As Nielsen et al. noted, a specific signal pathway by which granulin activates hStCs into αSMA+-MFs should be determined because there have been no reports regarding the activation of hStCs stimulated with granulin and the cell-surface receptor for granulin remains undetermined. Third, how does periostin contribute to metastatic PDAC growth in the liver? Recent studies have demonstrated that resident lung fibroblasts induce periostin and another ECM protein, tenascin, in breast cancer metastasis.6, 7 Periostin and tenascin enhance the colonization of breast cancer cells in the lungs by promoting Wnt and Wnt/Notch signaling, respectively, and periostin-deficient mice exhibited approximately 90% reduction in lung metastasis compared to wild-type mice. These studies suggest that fibroblast-derived periostin and tenascin create the metastatic tumor environment and stimulate the Wnt and Notch signaling pathways, thereby leading to cancer cell growth. Fourth, what is the role of resident KCs in PDAC metastasis? Although Nielsen et al. argued that KCs are primarily involved in the initial tumor cell seeding and are no longer required for PDAC outgrowth at the metastatic sites, Costa-Silva et al. demonstrated that PDAC-derived exosomes enriched with migration inhibitory factor promote KC activation and subsequent premetastatic niche formation in the liver.8 Thus, the role of liver-resident KCs in PDAC metastasis and outgrowth requires further evaluation. In summary, the findings reported herein underscore the importance of granulin and periostin in PDAC liver metastasis and provide insight into the role of the tumor microenvironment consisting of MAMs and hStC-derived MFs. A molecular approach can be used to develop novel drug targets against the outgrowth of metastatic PDAC. In addition, granulin, which is highly expressed in circulating IMs of PDAC patients, exhibits potential as a prognostic marker for liver metastasis. Misako Sato-Matsubara, Ph.D. Norifumi Kawada, M.D., Ph.D. Department of Hepatology Graduate School of Medicine Osaka City University Osaka, Japan