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Transforming growth factor beta (TGFβ) activity is linked to metastasis in many cancer types, but whether TGFβ activity is necessary for squamous cell carcinoma (SCC) lung metastasis has not been studied. Here we used a lung metastatic SCC model derived from keratin 15 (K15). KrasG12D.Smad4-/- SCC and human SCC specimens to identify metastasis drivers and test therapeutic interventions. We demonstrated that a TGFβ receptor (TGFβR) inhibitor reduced lung metastasis in mouse SCC correlating with reduced CD11b+/Ly6G+ myeloid cells positive for inducible nitric oxide synthase (iNOS). Further, TGFβ activity and iNOS were higher in primary human oral SCCs with metastasis than SCCs without metastasis. Consistently, either depleting myeloid cells with anti-Gr1 antibody or inhibiting iNOS with L-N6-(1-iminoethyl)-l-lysine (L-NIL) reduced SCC lung metastasis. L-NIL treated tumor-bearing mice exhibited reductions in tumor-infiltrating myeloid cells and in plasma Cxcl5 levels, and attenuated primary tumor growth with increased apoptosis and decreased proliferation. Blocking Cxcl5 with an antagonist of its receptor Cxcr2, SB225002, also reduced SCC lung metastasis.

Transforming growth factor beta (TGFβ) and programmed death-ligand 1 (PD-L1) are often overproduced in refractory squamous cell carcinoma (SCC). We examined spatial patterns of PD-L1+ cells in mouse and human SCCs and found that PD-L1 was primarily expressed on infiltrating leukocytes. Although combined TGFβ and PD-L1 blockade are undergoing cancer clinical trials, there are no predictive markers for therapeutic responders. To address this, we used both a small molecule TGFβ inhibitor in combination with anti-PD-L1 and a bifunctional fusion protein targeting both TGFβ and PD-L1 to treat mouse SCCs and found TGFβ inhibition enhanced PD-L1 blockade-induced tumor eradication in multiple tumor models. Furthermore, we identified distinct cell populations of responders and non-responders to bintrafusp alfa, with responders showing a shift toward a more immune-permissive microenvironment. The cellular and molecular signatures of responders versus non-responders to combined TGFβ and PD-L1 blockade provide important insights into future personalized immunotherapy in SCC.

Tumor-associated macrophages (TAM) in the tumor microenvironment (TME) cooperate with cancer stem cells (CSC) to maintain stemness. We recently identified cluster of differentiation 44 (CD44) as a surface marker defining head and neck squamous cell carcinoma (HNSCC) CSC. PI3K-4EBP1-SOX2 activation and signaling regulate CSC properties, yet the upstream molecular control of this pathway and the mechanisms underlying cross-talk between TAM and CSC in HNSCC remain largely unknown. Because CD44 is a molecular mediator in the TME, we propose here that TAM-influenced CD44 signaling could mediate stemness via the PI3K-4EBP1-SOX2 pathway, possibly by modulating availability of hyaluronic acid (HA), the main CD44 ligand. HNSCC IHC was used to identify TAM/CSC relationships, and in vitro coculture spheroid models and in vivo mouse models were used to identify the influence of TAMs on CSC function via CD44. Patient HNSCC-derived TAMs were positively and negatively associated with CSC marker expression at noninvasive and invasive edge regions, respectively. TAMs increased availability of HA and increased cancer cell invasion. HA binding to CD44 increased PI3K-4EBP1-SOX2 signaling and the CSC fraction, whereas CD44-VCAM-1 binding promoted invasive signaling by ezrin/PI3K. In vivo, targeting CD44 decreased PI3K-4EBP1-SOX2 signaling, tumor growth, and CSC. TAM depletion in syngeneic and humanized mouse models also diminished growth and CSC numbers. Finally, a CD44 isoform switch regulated epithelial-to-mesenchymal plasticity as standard form of CD44 and CD44v8-10 determined invasive and tumorigenic phenotypes, respectively. We have established a mechanistic link between TAMs and CSCs in HNSCC that is mediated by CD44 intracellular signaling in response to extracellular signals. SIGNIFICANCE: These findings establish a mechanistic link between tumor cell CD44, TAM, and CSC properties at the tumor-stroma interface that can serve as a vital area of focus for target and drug discovery.

PurposeSMAD4 loss causes genomic instability and the initiation/progression of head and neck squamous cell carcinoma (HNSCC). Here, we study whether SMAD4 loss sensitizes HNSCCs to olaparib (PARP inhibitor) in combination with radiotherapy (RT).Experimental designWe analyzed HNSCC The Cancer Genome Atlas data for SMAD4 expression in association with FANC/BRCA family gene expression. Human HNSCC cell lines were screened for sensitivity to olaparib. Isogenic HNSCC cell lines were generated to restore or reduce SMAD4 expression and treated with olaparib, radiation, or the combination. HNSCC pretreatment specimens from a phase I trial investigating olaparib were analyzed.ResultsSMAD4 levels correlated with levels of FANC/BRCA genes in HNSCC. HNSCC cell lines with SMAD4 homozygous deletion were sensitive to olaparib. In vivo, olaparib or RT monotherapy reduced tumor volumes in SMAD4-mutant but not SMAD4-positive tumors. Olaparib with RT dual therapy sustained tumor volume reduction in SMAD4-deficient (mutant or knockdown) xenografts, which exhibited increased DNA damage and cell death compared with vehicle-treated tumors. In vitro, olaparib alone or in combination with radiation caused lower clonogenic survival, more DNA damage-associated cell death, and less proliferation in SMAD4-deficient cells than in SMAD4-positive (endogenous SMAD4 or transduced SMAD4) cells. Applicable to clinic, 5 out of 6 SMAD4-negative HNSCCs and 4 out of 8 SMAD4-positive HNSCCs responded to a standard treatment plus olaparib in a phase I clinical trial, and SMAD4 protein levels inversely correlated with DNA damage.ConclusionsSMAD4 levels are causal in determining sensitivity to PARP inhibition in combination with RT in HNSCCs.

SMAD4 is a potent tumor suppressor and a central mediator of the TGFß signaling pathway. SMAD4 genetic loss is frequent in squamous cell carcinomas (SCCs). Reports of SMAD4 expression in SCCs vary significantly possibly due to inter-tumor heterogeneity or technical reasons. SMAD4 loss is an initiation event for SCCs. In tumor epithelial cells, SMAD4 loss causes increased proliferation, decreased apoptosis, and "Brca-like" genomic instability associated with DNA repair defects. SMAD4 loss also plays a role in the expansion of cancer stem cells. Epithelial SMAD4 loss causes overexpression of TGFß that is released into the tumor microenvironment and contributes to SCC progression through proinflammatory and immune evasive mechanisms. SMAD4 loss, while not a direct therapeutic target, is associated with multiple targetable pathways that require further therapeutic studies. Altogether, SMAD4 loss is a potential biomarker in SCCs that should be further studied for its values in prognostic and therapeutic predictions. Such information will potentially guide future biomarker-driven clinical trial designs and improve SCC patient outcomes.

UnlabelledNeoadjuvant chemotherapy (NAC) induces a pathologic complete response (pCR) in approximately 30% of patients with triple-negative breast cancers (TNBC). In patients lacking a pCR, NAC selects a subpopulation of chemotherapy-resistant tumor cells. To understand the molecular underpinnings driving treatment-resistant TNBCs, we performed comprehensive molecular analyses on the residual disease of 74 clinically defined TNBCs after NAC, including next-generation sequencing (NGS) on 20 matched pretreatment biopsies. Combined NGS and digital RNA expression analysis identified diverse molecular lesions and pathway activation in drug-resistant tumor cells. Ninety percent of the tumors contained a genetic alteration potentially treatable with a currently available targeted therapy. Thus, profiling residual TNBCs after NAC identifies targetable molecular lesions in the chemotherapy-resistant component of the tumor, which may mirror micrometastases destined to recur clinically. These data can guide biomarker-driven adjuvant studies targeting these micrometastases to improve the outcome of patients with TNBC who do not respond completely to NAC.SignificanceThis study demonstrates the spectrum of genomic alterations present in residual TNBC after NAC. Because TNBCs that do not achieve a CR after NAC are likely to recur as metastatic disease at variable times after surgery, these alterations may guide the selection of targeted therapies immediately after mastectomy before these metastases become evident.

Background: The importance of the mTOR complex 2 (mTORC2) signaling complex in tumor progression is becoming increasingly recognized. HER2-amplified breast cancers use Rictor/mTORC2 signaling to drive tumor formation, tumor cell survival and resistance to human epidermal growth factor receptor 2 (HER2)-targeted therapy. Cell motility, a key step in the metastatic process, can be activated by mTORC2 in luminal and triple negative breast cancer cell lines, but its role in promoting metastases from HER2-amplified breast cancers is not yet clear.Methods: Because Rictor is an obligate cofactor of mTORC2, we genetically engineered Rictor ablation or overexpression in mouse and human HER2-amplified breast cancer models for modulation of mTORC2 activity. Signaling through mTORC2-dependent pathways was also manipulated using pharmacological inhibitors of mTOR, Akt, and Rac. Signaling was assessed by western analysis and biochemical pull-down assays specific for Rac-GTP and for active Rac guanine nucleotide exchange factors (GEFs). Metastases were assessed from spontaneous tumors and from intravenously delivered tumor cells. Motility and invasion of cells was assessed using Matrigel-coated transwell assays.Results: We found that Rictor ablation potently impaired, while Rictor overexpression increased, metastasis in spontaneous and intravenously seeded models of HER2-overexpressing breast cancers. Additionally, migration and invasion of HER2-amplified human breast cancer cells was diminished in the absence of Rictor, or upon pharmacological mTOR kinase inhibition. Active Rac1 was required for Rictor-dependent invasion and motility, which rescued invasion/motility in Rictor depleted cells. Rictor/mTORC2-dependent dampening of the endogenous Rac1 inhibitor RhoGDI2, a factor that correlated directly with increased overall survival in HER2-amplified breast cancer patients, promoted Rac1 activity and tumor cell invasion/migration. The mTORC2 substrate Akt did not affect RhoGDI2 dampening, but partially increased Rac1 activity through the Rac-GEF Tiam1, thus partially rescuing cell invasion/motility. The mTORC2 effector protein kinase C (PKC)α did rescue Rictor-mediated RhoGDI2 downregulation, partially rescuing Rac-guanosine triphosphate (GTP) and migration/motility.Conclusion: These findings suggest that mTORC2 uses two coordinated pathways to activate cell invasion/motility, both of which converge on Rac1. Akt signaling activates Rac1 through the Rac-GEF Tiam1, while PKC signaling dampens expression of the endogenous Rac1 inhibitor, RhoGDI2. [ABSTRACT FROM AUTHOR]

Squamous cell carcinoma (SCC) is the second commonest type of skin cancer, and SCCs make up about 90% of head and neck cancers (HNSCCs). HNSCCs harbor two frequent molecular alterations, namely, gain-of-function alterations of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha ( PIK3CA) and loss-of-function mutations of tumor protein p53 ( TP53 ). However, it remains poorly understood whether HNSCCs harboring different genetic alterations exhibit differential immune tumor microenvironments (TME). It also remains unknown whether PIK3CA hyperactivation and TP53 deletion can lead to SCC development spontaneously. Here, we analyzed the Cancer Genome Atlas (TCGA) datasets of HNSCCs and found that patients with both PIK3CA and TP53 alterations exhibited worse survival, significantly lower CD8 tumor infiltrating lymphocytes (TILs) and higher M0 macrophages than other controls. To better model human tumorigenesis, we deleted TP53 and constitutively activated PIK3CA in mouse keratin-15-expressing stem cells, which leads to the spontaneous development of multilineage tumors including SCCs, termed Keratin-15-p53-PIK3CA (KPPA) tumors. KPPA tumors were heavily infiltrated with myeloid-derived suppressor cells (MDSCs), with a drastically increased ratio of polymorphonuclear-MDSC (PMN-MDSC) versus monocytic-MDSC (M-MDSC). CD8 TILs expressed more PD-1 and reduced their polyfunctionality. Overall, we established a genetic model to mimic human HNSCC pathogenesis, manifested with an immunosuppressive TME, which may help further elucidate immune evasion mechanisms and develop more effective immunotherapies for HNSCCs.

Background: Plasticity of the ERBB receptor network has been suggested to cause acquired resistance to anti-human epidermal growth factor receptor 2 (HER2) therapies. Thus, we studied whether a novel approach using an ERBB1-3-neutralizing antibody mixture can block these compensatory mechanisms of resistance., Methods: HER2+ cell lines and xenografts (n ≥ 6 mice per group) were treated with the ERBB1-3 antibody mixture Pan-HER, trastuzumab/lapatinib (TL), trastuzumab/pertuzumab (TP), or T-DM1. Downregulation of ERBB receptors was assessed by immunoblot analysis and immunohistochemistry. Paired pre- and post-T-DM1 tumor biopsies from patients (n = 11) with HER2-amplified breast cancer were evaluated for HER2 and P-HER3 expression by immunohistochemistry and/or fluorescence in situ hybridization. ERBB ligands were measured by quantitative reverse transcription polymerase chain reaction. Drug-resistant cells were generated by chronic treatment with T-DM1. All statistical tests were two-sided., Results: Treatment with Pan-HER inhibited growth and promoted degradation of ERBB1-3 receptors in a panel of HER2+ breast cancer cells. Compared with TL, TP, and T-DM1, Pan-HER induced a similar antitumor effect against established BT474 and HCC1954 tumors, but was superior to TL against MDA-361 xenografts (TL mean = 2026 mm 3 , SD = 924 mm 3 , vs Pan-HER mean = 565 mm 3 , SD = 499 mm 3 , P = .04). Pan-HER-treated BT474 xenografts did not recur after treatment discontinuation, whereas tumors treated with TL, TP, and T-DM1 did. Post-TP and post-T-DM1 recurrent tumors expressed higher levels of neuregulin-1 (NRG1), HER3 and P-HER3 (all P < .05). Higher levels of P-HER3 protein and NRG1 mRNA were also observed in HER2+ breast cancers progressing after T-DM1 and trastuzumab (NRG1 transcript fold change ± SD; pretreatment = 2, SD = 1.9, vs post-treatment = 11.4, SD = 10.3, P = .04). The HER3-neutralizing antibody LJM716 resensitized the drug-resistant cells to T-DM1, suggesting a causal association between the NRG1-HER3 axis and drug resistance. Finally, Pan-HER treatment inhibited growth of HR6 trastuzumab- and T-DM1-resistant xenografts., Conclusions: These data suggest that upregulation of a NRG1-HER3 axis can mediate escape from anti-HER2 therapies. Further, multitargeted antibody mixtures, such as Pan-HER, can simultaneously remove and/or block targeted ERBB receptor and ligands, thus representing an effective approach against drug-sensitive and -resistant HER2+ cancers., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Purpose: FGFR1 amplification occurs in approximately 15% of estrogen receptor-positive (ER + ) human breast cancers. We investigated mechanisms by which FGFR1 amplification confers antiestrogen resistance to ER + breast cancer. Experimental Design: ER + tumors from patients treated with letrozole before surgery were subjected to Ki67 IHC, FGFR1 FISH, and RNA sequencing (RNA-seq). ER + / FGFR1 -amplified breast cancer cells, and patient-derived xenografts (PDX) were treated with FGFR1 siRNA or the FGFR tyrosine kinase inhibitor lucitanib. Endpoints were cell/xenograft growth, FGFR1/ERα association by coimmunoprecipitation and proximity ligation, ER genomic activity by ChIP sequencing, and gene expression by RT-PCR. Results: ER + / FGFR1 -amplified tumors in patients treated with letrozole maintained cell proliferation (Ki67). Estrogen deprivation increased total and nuclear FGFR1 and FGF ligands expression in ER + / FGFR1- amplified primary tumors and breast cancer cells. In estrogen-free conditions, FGFR1 associated with ERα in tumor cell nuclei and regulated the transcription of ER-dependent genes. This association was inhibited by a kinase-dead FGFR1 mutant and by treatment with lucitanib. ChIP-seq analysis of estrogen-deprived ER + / FGFR1 -amplified cells showed binding of FGFR1 and ERα to DNA. Treatment with fulvestrant and/or lucitanib reduced FGFR1 and ERα binding to DNA. RNA-seq data from FGFR1 -amplified patients' tumors treated with letrozole showed enrichment of estrogen response and E2F target genes. Finally, growth of ER + / FGFR1- amplified cells and PDXs was more potently inhibited by fulvestrant and lucitanib combined than each drug alone. Conclusion s : These data suggest the ERα pathway remains active in estrogen-deprived ER + / FGFR1 -amplified breast cancers. Therefore, these tumors are endocrine resistant and should be candidates for treatment with combinations of ER and FGFR antagonists. Clin Cancer Res; 23(20); 6138-50. ©2017 AACR ., (©2017 American Association for Cancer Research.)