Junttila and colleagues evaluate chemotherapeutic drugs conjugated to antibodies (ADC) that target the intestinal stem cell marker LGR5, which is highly enriched in intestinal tumors. They conjugated two distinct chemotherapeutics to this antibody using a linker designed to liberate the drug upon internalization of the antibody. Both ADCs attenuated growth of xenograft cancer models. One also impacted normal intestinal function. While administration of the least gastrointestinal toxic ADC slowed proliferation in a novel genetically engineered mouse model of colorectal cancer, tumor numbers and size showed little change initially. Over a longer time frame however, this decreased proliferation led to smaller tumors and improved survival compared to an unconjugated control antibody, consistent with specific targeting of the LGR5+ cancer stem cells (CSC). These results validate targeting CSC with ADC as a therapeutic strategy.

Junttila MR, Mao W, Wang X, Wang B–E, Pham T, Flygare J, et al. Targeting LGR5+ cells with an antibody-drug conjugate for the treatment of colon cancer. Sci Transl Med 2015;7:314ra186.

Hoshino and colleagues found that tumors preferentially metastasizing to different tissues secrete distinct exosome pools. Carcinoma cells disseminating preferentially to lungs secreted α6β4 integrin–positive exosomes, which became tethered to lung epithelial cells. Conversely, liver metastasizing cells secreted αvβ5 exosomes, with preferential affinity towards liver Kupffer cells. Strikingly, injection of liver-targeting exosomes was sufficient to redirect lung metastatic cells to disseminate preferentially to the liver. In the target tissue, exosome uptake stimulated SRC activation and secretion of proinflammatory S100 proteins, paving the way for metastasis. Importantly, integrin-positive exosomes could be detected from patient plasma, with high levels of exosomal integrins identifying patients that developed organ-specific metastasis. These data suggest that the seed-and-soil hypothesis of cancer spread needs to be refined, given that exosomes can precondition the soil before the seed arrives.

Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic Mark M, et al. Tumour exosome integrins determine organotropic metastasis. Nature 2015;527:329-35.

Zhang and colleagues analyzed gene expression and immunohistochemistry data from primary and metastatic breast cancer to brain, revealing a curious drop in PTEN expression in brain metastases. Exposing tumors to conditioned media from astrocytes resulted in a significant drop in PTEN mRNA and PTEN protein expression. Astrocyte-specific knock-out of the microRNA-17∼92 cluster, known to target PTEN, blocked downregulation of PTEN in B16BL6 melanoma cells and reduced tumor growth in the brain, an effect dependent on astrocyte-derived exosome-mediated transfer of miR-19 to tumor cells. Breast tumors metastatic to brain exhibited decreased PTEN, increased CCL2, and increased microglia/macrophages. Thus, decreased PTEN expression led to increased CCL2 expression (secondary to decreased phosphorylation of NF-κB), resulting in myeloid cell recruitment and tumor outgrowth in the brain.

Zhang L, Zhang S, Yao J, Lowery FJ, Zhang Q, Huang WC, et al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature 2015;527:100-4.

MLK4, a member of the mixed-lineage family of kinases, is persistently mutated in a range of malignancies. Marusiak and colleagues evaluated the functional consequences of MLK4 mutations in colon cancer. A majority were loss-of-function mutations that had a dominant negative effect. The authors identified a new MLK4 catalytic domain structure. Restoring expression of MLK4 in cells harboring MLK4-inactivating mutations resulted in reduced in vitro tumor attributes: cell viability, proliferation, and colony formation as well as delayed tumor growth in vivo. The authors further provided mechanistic insight into the signaling that occurs following restoration of MLK4 expression and determined that MLK4 induced the JNK pathway and its downstream targets. Thus, the authors identified MLK4 as a novel colon cancer tumor suppressing kinase that is frequently mutated in colon cancer.

Marusiak AA, Stephenson NL, Baik H, Trotter EW, Li Y, Blyth K, et al. Recurrent MLK4 loss-of-function mutations suppress JNK signaling to promote colon tumorigenesis. Cancer Research; Published OnlineFirst December 4, 2015; doi:10.1158/0008-5472.CAN-15-0701-T.

Chimeric antigen receptor (CAR)-armed T cells targeting the CD19 antigen (CART-19) are highly effective in CD19-positive B-cell acute lymphoblastic leukemias. Sotillo and colleagues describe molecular events in CD19 associated with immune escape after CAR-T cell therapy, demonstrating loss of expression, genetic alterations, and alternative splicing lacking exon 2, leading to decreased levels of full-length CD19. The novel spliced CD19 isoform partly rescued functional defects associated with complete loss of CD19 expression in vitro, also losing the CD19 epitope necessary for CART-19 recognition. SRSF3 was identified as a splicing factor involved in exon 2 retention, and in samples from two pediatric patients with CD19-negative relapses, levels of SRSF3 decreased at relapse. This study suggests that strategies to overcome the outgrowth of antigen loss in tumor cells may improve efficacy and durability in patients.

Sotillo E, Barrett DM, Black KL, Bagashev A, Oldridge D, Wu G, et al. Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy. Cancer Discov 2015;5:1282-95.

A characteristic of solid tumors is the presence of activated fibroblasts (cancer associated fibroblasts, CAF) and increased matrix deposition in tumor stroma. Increased contractility of CAFs facilitates metastasis and contributes to proliferation via stiffening of the tumor microenvironment. Madsen and colleagues found that CAF activation is reversible. Chronic hypoxia deactivated CAFs, impairing their ability to model the tumor stroma to enable cancer cell invasion. Mechanistically, hypoxia reverted CAFs via inhibition of prolyl hydroxylase domain protein 2 (PHD2) and subsequent stabilization of HIF1α and its downstream targets. Mimicking hypoxia, depletion of PHD2 in CAFs failed to induce metastasis when coinjected with cancer cells. Furthermore, pharmacological inhibition of PHD2 inhibited spontaneous metastasis in vivo. Given that most solid tumors have activated stroma, these data indicate the potential of healthy tissue as a cancer barrier.

Madsen CD, Pedersen JT, Venning FA, Singh LB, Moeendarbary E, Charras G, et al. Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis. EMBO Rep 2015;16:1394-408.

Note: Breaking Advances are written by Cancer Research editors. Readers are encouraged to consult the articles referred to in each item for full details on the findings described.