Therapeutic Targeting of LIF Overcomes Macrophage-mediated Immunosuppression of the Local Tumor Microenvironment.

PURPOSE: Leukemia inhibitory factor (LIF) is a multifunctional cytokine with numerous reported roles in cancer and is thought to drive tumor development and progression. Characterization of LIF and clinical-stage LIF inhibitors would increase our understanding of LIF as a therapeutic target. EXPERIMENTAL DESIGN: We first tested the association of LIF expression with transcript signatures representing multiple processes regulating tumor development and progression. Next, we developed MSC-1, a high-affinity therapeutic antibody that potently inhibits LIF signaling and tested it in immune competent animal models of cancer. RESULTS: LIF was associated with signatures of tumor-associated macrophages (TAM) across 7,769 tumor samples spanning 22 solid tumor indications. In human tumors, LIF receptor was highly expressed within the macrophage compartment and LIF treatment drove macrophages to acquire immunosuppressive capacity. MSC-1 potently inhibited LIF signaling by binding an epitope that overlaps with the gp130 receptor binding site on LIF. MSC-1 showed monotherapy efficacy in vivo and drove TAMs to acquire antitumor and proinflammatory function in syngeneic colon cancer mouse models. Combining MSC-1 with anti-PD1 leads to strong antitumor response and a long-term tumor-free survival in a significant proportion of treated mice. CONCLUSIONS: Overall, our findings highlight LIF as a therapeutic target for cancer immunotherapy.

Noncyclam tetraamines inhibit CXC chemokine receptor type 4 and target glioma-initiating cells.

The three stereoisomers of the noncyclam compound 1 (1(R,R), 1(S,S), and the meso form 1(S,R)) and their corresponding tetrahydrochlorides 11 were prepared from (S)- and (R)-2-methylpiperidine. We have evaluated their inhibitory activity on the CXC chemokine receptor type 4 (CXCR4), toxicity properties, and assessment of their effect on glioma initiating cells (GICs) in comparison with the prototype compound AMD3100. The IC(50) values determined on human recombinant (CHO) cells showed very similar inhibitory activities albeit a lower K(B) for AMD3100, with the 1(R,R) isomer being second in potency. All the compounds showed low cardiac toxicity but, contrary to AMD3100, gave maximum nonlethal doses of around 2.0 mg/kg. The CXCR4 inhibitors had an effect on the state of differentiation of GICs, decreasing the percentage of CD44+ cells in glioblastoma multiform neurospheres in vitro. Moreover, these CXCR4 inhibitors blocked the capacity of cells to initiate orthotopic tumors in immunocompromised mice.

TGF-ß Receptor Inhibitors Target the CD44(high)/Id1(high) Glioma-Initiating Cell Population in Human Glioblastoma.

Glioma-initiating cells (GICs), also called glioma stem cells, are responsible for tumor initiation, relapse, and therapeutic resistance. Here, we show that TGF-ß inhibitors, currently under clinical development, target the GIC compartment in human glioblastoma (GBM) patients. Using patient-derived specimens, we have determined the gene responses to TGF-ß inhibition, which include inhibitors of DNA-binding protein (Id)-1 and -3 transcription factors. We have identified a cell population enriched for GICs that expresses high levels of CD44 and Id1 and tend to be located in a perivascular niche. The inhibition of the TGF-ß pathway decreases the CD44(high)/Id1(high) GIC population through the repression of Id1 and Id3 levels, therefore inhibiting the capacity of cells to initiate tumors. High CD44 and Id1 levels confer poor prognosis in GBM patients.

TGF-beta increases glioma-initiating cell self-renewal through the induction of LIF in human glioblastoma.

Glioma-initiating cells (GICs) are responsible for the initiation and recurrence of gliomas. Here, we identify a molecular mechanism that regulates the self-renewal capacity of patient-derived GICs. We show that TGF-beta and LIF induce the self-renewal capacity and prevent the differentiation of GICs. TGF-beta induces the self-renewal capacity of GICs, but not of normal human neuroprogenitors, through the Smad-dependent induction of LIF and the subsequent activation of the JAK-STAT pathway. The effect of TGF-beta and LIF on GICs promotes oncogenesis in vivo. Some human gliomas express high levels of LIF that correlate with high expression of TGF-beta2 and neuroprogenitor cell markers. Our results show that TGF-beta and LIF have an essential role in the regulation of GICs in human glioblastoma.

Expression of p95HER2, a truncated form of the HER2 receptor, and response to anti-HER2 therapies in breast cancer.

BACKGROUND: Women with HER2-overexpressing breast cancers have poor prognosis, and many are resistant to the HER2 monoclonal antibody trastuzumab. A subgroup of HER2-overexpressing tumors also express p95HER2, an amino terminally truncated receptor that has kinase activity. Because p95HER2 cannot bind to trastuzumab but should be responsive to the HER2 tyrosine kinase inhibitor lapatinib, we compared the sensitivity of tumors expressing p95HER2 and tumors expressing the full-length HER2 receptor to these agents. METHODS: MCF-7 and T47D breast cancer cells were stably transfected with either full-length HER2 or p95HER2. We studied the effects of trastuzumab and lapatinib on receptor signaling, cell proliferation, and the growth of xenograft tumors. A paraffin-based immunofluorescence assay was developed to study the association between p95HER2 expression and sensitivity to trastuzumab in patients with advanced breast cancer. All statistical tests were two-sided. RESULTS: Treatment of p95HER2-expressing cells with lapatinib inhibited p95HER2 phosphorylation, reduced downstream phosphorylation of Akt and mitogen-activated protein kinases, inhibited cell growth (MCF-7p95HER2 clones, lapatinib versus control, mean growth inhibition = 57.6% versus 22.6%, difference = 35%, 95% confidence interval [CI] = 22.5% to 47.3%; P<.001; T47Dp95HER2 clones, lapatinib versus control, mean growth inhibition = 36.8% versus 20%, difference = 16.8%, 95% CI = 11.3% to 22.3%, P<.001), and inhibited growth of MCF-7p95HER2 xenograft tumors (lapatinib versus control, mean = 288.8 versus 435 mm3, difference = 146.2 mm3, CI = 73.8 to 218.5 mm3, P = .002). By contrast, treatment with trastuzumab had no effect on any of these parameters. Of 46 patients with metastatic breast cancer who were treated with trastuzumab, only one of nine patients (11.1%) expressing p95HER2 responded to trastuzumab (with a partial response), whereas 19 of the 37 patients (51.4%) with tumors expressing full-length HER2 achieved either a complete (five patients) or a partial (14 patients) response (P = .029). CONCLUSIONS: Breast tumors that express p95HER2 are resistant to trastuzumab and may require alternative or additional anti-HER2-targeting strategies.

Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation.

The overactivation of the HERs, a family of tyrosine kinase receptors, leads to the development of cancer. Although the canonical view contemplates HER receptors restricted to the secretory and endocytic pathways, full-length HER1, HER2 and HER3 have been detected in the nucleoplasm. Furthermore, limited proteolysis of HER4 generates nuclear C-terminal fragments (CTFs). Using cells expressing a panel of deletion and point mutants, here we show that HER2 CTFs are generated by alternative initiation of translation from methionines located near the transmembrane domain of the full-length molecule. In vitro and in vivo, HER2 CTFs are found in the cytoplasm and nucleus. Expression of HER2 CTFs to levels similar to those found in human tumors induces the growth of breast cancer xenografts in nude mice. Tumors dependent on CTFs are sensitive to inhibitors of the kinase activity but do not respond to therapeutic antibodies against HER2. Thus, the kinase domain seems necessary for the activity of HER2 CTFs and the presence of these HER2 fragments could account for the resistance to treatment with antibodies.

ZD1839, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, induces the formation of inactive EGFR/HER2 and EGFR/HER3 heterodimers and prevents heregulin signaling in HER2-overexpressing breast cancer cells.

PURPOSE: ZD1839 is a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) that has shown clinical activity against EGFR-expressing tumors. Our aim was to explore the effects of ZD1839 in breast cancer cell lines expressing different levels of EGFR and the closely related HER2 receptor. EXPERIMENTAL DESIGN: We studied the growth-inhibitory effects of ZD1839 in a series of breast carcinoma cell lines. In HER2-overexpressing BT-474 breast cancer cells, we studied the effects of ZD1839 on cell growth and heterodimerization of receptors under basal and ligand-stimulated conditions. RESULTS: ZD1839 was an equally potent inhibitor of growth in breast cancer cells expressing high levels of EGFR and HER2. In BT-474 breast cancer cells, ZD1839 abolished EGF- and heregulin-induced activation of ErbB receptors and downstream signaling molecules. Because ZD1839 does not inhibit the HER2 tyrosine kinase in vitro, and because heregulin is a ligand that activates HER2 by binding to HER3 and HER4 but does not bind to the EGFR, our findings suggested that ZD1839 interfered with HER2 function in intact cells. Searching for mechanisms, we report that ZD1839 induces the formation of inactive unphosphorylated EGFR/HER2 and EGFR/HER3 heterodimers. Furthermore, ZD1839 completely abolishes basal and heregulin-induced formation of active phosphorylated HER2/HER3 heterodimers. CONCLUSIONS: ZD1839 inhibits the growth of HER2-overexpressing breast cancer cells, possibly by sequestration of HER2 and HER3 receptors in an inactive heterodimer configuration with the EGFR. Our findings suggest that there is a strong rationale to conduct clinical trials of ZD1839 in patients with HER2-overexpressing breast tumors.