Precociously disseminated cancer cells may seed quiescent sites of future metastasis if they can protect themselves from immune surveillance. However, there is little knowledge about how such sites might be achieved. Here, we present evidence that prostate cancer stem–like cells (CSC) can be found in histopathologically negative prostate draining lymph nodes (PDLN) in mice harboring oncogene-driven prostate intraepithelial neoplasia (mPIN). PDLN-derived CSCs were phenotypically and functionally identical to CSC obtained from mPIN lesions, but distinct from CSCs obtained from frank prostate tumors. CSC derived from either PDLN or mPIN used the extracellular matrix protein Tenascin-C (TNC) to inhibit T-cell receptor–dependent T-cell activation, proliferation, and cytokine production. Mechanistically, TNC interacted with α5β1 integrin on the cell surface of T cells, inhibiting reorganization of the actin-based cytoskeleton therein required for proper T-cell activation. CSC from both PDLN and mPIN lesions also expressed CXCR4 and migrated in response to its ligand CXCL12, which was overexpressed in PDLN upon mPIN development. CXCR4 was critical for the development of PDLN-derived CSC, as in vivo administration of CXCR4 inhibitors prevented establishment in PDLN of an immunosuppressive microenvironment. Taken together, our work establishes a pivotal role for TNC in tuning the local immune response to establish equilibrium between disseminated nodal CSC and the immune system. Cancer Res; 75(10); 1–14. ©2015 AACR.

Precociously disseminated cancer cells may seed quiescent sites of future metastasis if they can protect themselves from immune surveillance. However, there is little knowledge about how such sites might be achieved. Here we present evidence that prostate cancer stem-like cells (CSC) can be found in histopathologically negative prostate draining lymph nodes (PDLN) in mice harboring oncogene-driven prostate intraepithelial neoplasia (mPIN). PDLN-derived CSC were phenotypically and functionally identical to CSC obtained from mPIN lesions, but distinct from CSCs obtained from frank prostate tumors. CSC derived from either PDLN or mPIN used the extracellular matrix protein Tenascin-C (TNC) to inhibit T cell receptor-dependent T cell activation, proliferation and cytokine production. Mechanistically, TNC interacted with α5β1 integrin on the cell surface of T cells, inhibiting reorganization of the actin-based cytoskeleton therein required for proper T cell activation. CSC from both PDLN and mPIN lesions also expressed CXCR4 and migrated in response to its ligand CXCL12, which was overexpressed in PDLN upon mPIN development. CXCR4 was critical for the development of PDLN-derived CSC, as in vivo administration of CXCR4 inhibitors prevented establishment in PDLN of an immunosuppressive microenvironment. Taken together, our work establishes a pivotal role for TNC in tuning the local immune response to establish equilibrium between disseminated nodal CSC and the immune system.

Semaphorins play important regulatory roles in diverse processes such as axon guidance, angiogenesis, and immune responses. We find that semaphorin-3C (sema3C) induces the collapse of the cytoskeleton of lymphatic endothelial cells (LEC) in a neuropilin-2-, plexin-D1-, and plexin-A1–dependent manner, while most other semaphorins, including antiangiogenic semaphorins such as sema3A do not. Sema3C is cleaved, like other class-3 semaphorins, by furin-like pro-protein convertases (FPPC). Cleaved sema3C (p65-Sema3C) was unable to induce the collapse of the cytoskeleton of LEC. FPPC are strongly upregulated in tumor cells. In order to examine the effects of full-length sema3C on tumor progression, we therefore generated an active point mutated furin cleavage-resistant sema3C (FR-sema3C). FR-sema3C inhibited potently proliferation of LEC and to a lesser extent proliferation of human umbilical vein–derived endothelial cells. FR-sema3C also inhibited VEGF-C–induced phosphorylation of VEGFR-3, ERK1/2, and AKT. Expression of recombinant FR-sema3C in metastatic, triple-negative LM2-4 breast cancer cells did not affect their migration or proliferation in vitro. However, tumors derived from FR-sema3C–expressing LM2-4 cells implanted in mammary fat pads developed at a slower rate, contained a lower concentration of blood vessels and lymph vessels, and metastasized much less effectively to lymph nodes. Interestingly, p65-Sema3C, but not FR-sema3C, rendered A549 lung cancer cells resistant to serum deprivation, suggesting that previously reported protumorigenic activities of sema3C may be due to p65-Sema3C produced by tumor cells. Our observations suggest that FR-sema3C may be further developed into a novel antitumorigenic drug. Cancer Res; 75(11); 1–10. ©2015 AACR.

Semaphorins play important regulatory roles in diverse processes such as axon guidance, angiogenesis and immune responses. We find that semaphorin-3C (sema3C) induces the collapse of the cytoskeleton of lymphatic endothelial cells (LEC) in a neuropilin-2, plexin-D1 and plexin-A1 dependent manner, while most other semaphorins, including anti-angiogenic semaphorins such as sema3A do not. Sema3C is cleaved, like other class-3 semaphorins, by furin like pro-protein convertases (FPPC). Cleaved sema3C (p65-Sema3C) was unable to induce the collapse of the cytoskeleton of LEC. FPPC are strongly up-regulated in tumor cells. In order to examine the effects of full length sema3C on tumor progression we therefore generated an active point mutated furin cleavage resistant sema3C (FR-sema3C). FR-sema3C inhibited potently proliferation of LEC and to a lesser extent proliferation of umbilical vein derived endothelial cells (HUVEC). FR-sema3C also inhibited VEGF-C induced phosphorylation of VEGFR-3, ERK1/2 and AKT. Expression of recombinant FR-sema3C in metastatic, triple negative LM2-4 breast cancer cells did not affect their migration or proliferation in-vitro. However, tumors derived from FR-sema3C expressing LM2-4 cells implanted in mammary fat pads developed at a slower rate, contained a lower concentration of blood vessels and lymph vessels, and metastasized much less effectively to lymph nodes. Interestingly, p65-Sema3C, but not FR-sema3C, rendered A549 lung cancer cells resistant to serum deprivation suggesting that previously reported pro-tumorigenic activities of sema3C may be due to p65-Sema3C produced by tumor cells. Our observations suggest that FR-sema3C may be further developed into a novel anti-tumorigenic drug.

Aneuploidy is frequently detected in human cancers and is implicated in carcinogenesis. Pharmacologic targeting of aneuploidy is an attractive therapeutic strategy, as this would preferentially eliminate malignant over normal cells. We previously discovered that CDK2 inhibition causes lung cancer cells with more than two centrosomes to undergo multipolar cell division leading to apoptosis, defined as anaphase catastrophe. Cells with activating KRAS mutations were especially sensitive to CDK2 inhibition. Mechanisms of CDK2-mediated anaphase catastrophe and how activated KRAS enhances this effect were investigated. Live-cell imaging provided direct evidence that following CDK2 inhibition, lung cancer cells develop multipolar anaphase and undergo multipolar cell division with the resulting progeny apoptotic. The siRNA-mediated repression of the CDK2 target and centrosome protein CP110 induced anaphase catastrophe of lung cancer cells. In contrast, CP110 overexpression antagonized CDK2 inhibitor–mediated anaphase catastrophe. Furthermore, activated KRAS mutations sensitized lung cancer cells to CDK2 inhibition by deregulating CP110 expression. Thus, CP110 is a critical mediator of CDK2 inhibition–driven anaphase catastrophe. Independent examination of murine and human paired normal–malignant lung tissues revealed marked upregulation of CP110 in malignant versus normal lung. Human lung cancers with KRAS mutations had significantly lower CP110 expression as compared with KRAS wild-type cancers. Thus, a direct link was found between CP110 and CDK2 inhibitor antineoplastic response. CP110 plays a mechanistic role in response of lung cancer cells to CDK2 inhibition, especially in the presence of activated KRAS mutations. Cancer Res; 75(10); 1–10. ©2015 AACR.