Neil E. Bhola

TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer

After an initial response to chemotherapy, many patients with triple-negative breast cancer (TNBC) have recurrence of drug-resistant metastatic disease. Studies with TNBC cells suggest that chemotherapy-resistant populations of cancer stem-like cells (CSCs) with self-renewing and tumor-initiating capacities are responsible for these relapses. TGF-β has been shown to increase stem-like properties in human breast cancer cells. We analyzed RNA expression in matched pairs of primary breast cancer biopsies before and after chemotherapy. Biopsies after chemotherapy displayed increased RNA transcripts of genes associated with CSCs and TGF-β signaling. In TNBC cell lines and mouse xenografts, the chemotherapeutic drug paclitaxel increased autocrine TGF-β signaling and IL-8 expression and enriched for CSCs, as indicated by mammosphere formation and CSC markers. The TGF-β type I receptor kinase inhibitor LY2157299, a neutralizing TGF-β type II receptor antibody, and SMAD4 siRNA all blocked paclitaxel-induced IL8 transcription and CSC expansion. Moreover, treatment of TNBC xenografts with LY2157299 prevented reestablishment of tumors after paclitaxel treatment. These data suggest that chemotherapy-induced TGF-β signaling enhances tumor recurrence through IL-8-dependent expansion of CSCs and that TGF-β pathway inhibitors prevent the development of drug-resistant CSCs. These findings support testing a combination of TGF-β inhibitors and anticancer chemotherapy in patients with TNBC.

Cross-talk between G Protein–Coupled Receptor and Epidermal Growth Factor Receptor Signaling Pathways Contributes to Growth and Invasion of Head and Neck Squamous Cell Carcinoma

G protein-coupled receptors (GPCR) and the epidermal growth factor receptor (EGFR) are often both overexpressed and contribute to the growth of cancers by activating autocrine pathways. GPCR ligands have been reported to trigger EGFR signaling via receptor cross-talk in cancer cells. Here, we show that GPCR ligands prostaglandin E2 (PGE2) and bradykinin (BK) activate EGFR signaling. Inhibition of EGFR using several strategies, including small-molecule inhibitors and an EGFR-specific antibody, resulted in partial attenuation of signaling downstream of EGFR. PGE2 and BK triggered EGFR signaling by increasing selective autocrine release of transforming growth factor-alpha (TGF-alpha). Inhibition of tumor necrosis factor-alpha-converting enzyme abrogated BK- or PGE2-mediated activation of EGFR signaling. Both PGE2 and BK stimulated head and neck squamous cell carcinoma (HNSCC) invasion via EGFR. Treatment of HNSCC cells with the BK antagonist CU201 resulted in growth inhibition. The combination of CU201 with the EGFR small-molecule inhibitor erlotinib resulted in additive inhibitory effects on HNSCC cell growth in vitro. Inhibition of the PGE2 synthesis pathway with sulindac induced HNSCC cytotoxicity at high doses (EC(50), 620 micromol/L). However, combined inhibition of both EGFR with the tyrosine kinase inhibitor erlotinib and GPCR with sulindac at low doses of 6 and 310 micromol/L, respectively, resulted in synergistic killing of HNSCC tumor cells. Combined blockade of both EGFR and GPCRs may be a rational strategy to treat cancers, including HNSCC that shows cross-talk between GPCR and EGFR signaling pathways.

Profiling of residual breast cancers after neoadjuvant chemotherapy identifies DUSP4 deficiency as a mechanism of drug resistance

Neoadjuvant chemotherapy (NAC) induces a pathological complete response (pCR) in ∼30% of patients with breast cancer. However, many patients have residual cancer after chemotherapy, which correlates with a higher risk of metastatic recurrence and poorer outcome than those who achieve a pCR. We hypothesized that molecular profiling of tumors after NAC would identify genes associated with drug resistance. Digital transcript counting was used to profile surgically resected breast cancers after NAC. Low concentrations of dual specificity protein phosphatase 4 (DUSP4), an ERK phosphatase, correlated with high post-NAC tumor cell proliferation and with basal-like breast cancer (BLBC) status. BLBC had higher DUSP4 promoter methylation and gene expression patterns of Ras-ERK pathway activation relative to other breast cancer subtypes. DUSP4 overexpression increased chemotherapy-induced apoptosis, whereas DUSP4 depletion dampened the response to chemotherapy. Reduced DUSP4 expression in primary tumors after NAC was associated with treatment-refractory high Ki-67 scores and shorter recurrence-free survival. Finally, inhibition of mitogen-activated protein kinase kinase (MEK) synergized with docetaxel treatment in BLBC xenografts. Thus, DUSP4 downregulation activates the Ras-ERK pathway in BLBC, resulting in an attenuated response to anti-cancer chemotherapy.

Trastuzumab-Resistant Cells Rely on a HER2-PI3K-FoxO-Survivin Axis and Are Sensitive to PI3K Inhibitors

The antibody trastuzumab is approved for treatment of patients with HER2 (ERBB2)-overexpressing breast cancer. A significant fraction of these tumors are either intrinsically resistant or acquire resistance rendering the drug ineffective. The development of resistance has been attributed to failure of the antibody to inhibit phosphoinositide 3-kinase (PI3K), which is activated by the HER2 network. Herein, we examined the effects of PI3K blockade in trastuzumab-resistant breast cancer cell lines. Treatment with the pan-PI3K inhibitor XL147 and trastuzumab reduced proliferation and pAKT levels, triggering apoptosis of trastuzumab-resistant cells. Compared with XL147 alone, the combination exhibited a superior antitumor effect against trastuzumab-resistant tumor xenografts. Furthermore, treatment with XL147 and trastuzumab reduced the cancer stem-cell (CSC) fraction within trastuzumab-resistant cells both in vitro and in vivo. These effects were associated with FoxO-mediated inhibition of transcription of the antiapoptosis gene survivin (BIRC5) and the CSC-associated cytokine interleukin-8. RNA interference-mediated or pharmacologic inhibition of survivin restored sensitivity to trastuzumab in resistant cells. In a cohort of patients with HER2-overexpressing breast cancer treated with trastuzumab, higher pretreatment tumor levels of survivin RNA correlated with poor response to therapy. Together, our results suggest that survivin blockade is required for therapeutic responses to trastuzumab and that by combining trastuzumab and PI3K inhibitors, CSCs can be reduced within HER2(+) tumors, potentially preventing acquired resistance to anti-HER2 therapy.

Antitumor mechanisms of combined gastrin-releasing peptide receptor and epidermal growth factor receptor targeting in head and neck cancer

Head and neck squamous cell carcinoma (HNSCC) is characterized by epidermal growth factor receptor (EGFR) overexpression, where EGFR levels correlate with survival. To date, EGFR targeting has shown limited antitumor effects in head and neck cancer when administrated as monotherapy. We previously identified a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) aurocrine regulatory pathway in HNSCC, where GRP stimulates Src-dependent cleavage of EGFR proligands with subsequent EGFR phosphorylation and mitogen-activated protein kinase (MAPK) activation. To determine whether GRPR targeting can enhance the antitumor efficacy of EGFR inhibition, we investigated the effects of a GRPR antagonist (PD176252) in conjunction with an EGFR tyrosine kinase inhibitor (erlotinib). Combined blockade of GRPR and EGFR pathways significantly inhibited HNSCC, but not immortalized mucosal epithelial cell, proliferation, invasion, and colony formation. In addition, the percentage of apoptotic cells increased upon combined inhibition. The enhanced antitumor efficacy was accompanied by increased expression of cleaved poly(ADP-ribose) polymerase (PARP) and decreased phospho-EGFR, phospho-MAPK, and proliferating cell nuclear antigen (PCNA). Using reverse-phase protein microarray (RPPA), we further detected decreased expression of phospho–c-Jun, phospho-p70S6K, and phospho-p38 with combined targeting. Cumulatively, these results suggest that GRPR targeting can enhance the antitumor effects of EGFR inhibitors in head and neck cancer. [Mol Cancer Ther 2007;6(4):1414–24]

Activation of MAPK pathways due to DUSP4 loss promotes cancer stem cell-like phenotypes in basal-like breast cancer

Basal-like breast cancer (BLBC) is an aggressive disease that lacks a clinically approved targeted therapy. Traditional chemotherapy is effective in BLBC, but it spares the cancer stem cell (CSC)-like population, which is likely to contribute to cancer recurrence after the initial treatment. Dual specificity phosphatase-4 (DUSP4) is a negative regulator of the mitogen-activated protein kinase (MAPK) pathway that is deficient in highly aggressive BLBCs treated with chemotherapy, leading to aberrant MAPK activation and resistance to taxane-induced apoptosis. Herein, we investigated how DUSP4 regulates the MAP-ERK kinase (MEK) and c-jun-NH2-kinase (JNK) pathways in modifying CSC-like behavior. DUSP4 loss increased mammosphere formation and the expression of the CSC-promoting cytokines interleukin (IL)-6 and IL-8. These effects were caused in part by loss of control of the MEK and JNK pathways and involved downstream activation of the ETS-1 and c-JUN transcription factors. Enforced expression of DUSP4 reduced the CD44(+)/CD24(-) population in multiple BLBC cell lines in a MEK-dependent manner, limiting tumor formation of claudin-low SUM159PT cells in mice. Our findings support the evaluation of MEK and JNK pathway inhibitors as therapeutic agents in BLBC to eliminate the CSC population.

Honokiol inhibits epidermal growth factor receptor signaling and enhances the antitumor effects of epidermal growth factor receptor inhibitors.

Purpose: This study aimed to investigate the utility of honokiol, a naturally occurring compound, in the treatment of head and neck squamous cell carcinoma (HNSCC) as well as its ability to target the epidermal growth factor receptor (EGFR), a critical therapeutic target in HNSCC, and to enhance the effects of other EGFR-targeting therapies. Experimental Design: Human HNSCC cell lines and the xenograft animal model of HNSCC were used to test the effects of honokiol treatment. Results: Honokiol was found to inhibit growth in human HNSCC cell lines, with 50% effective concentration (EC50) values ranging from 3.3 to 7.4 μmol/L, and to induce apoptosis, as shown through Annexin V staining. These effects were associated with inhibition of EGFR signaling, including downstream inhibition of mitogen-activated protein kinase, Akt, and signal transducer and activator of transcription 3 (STAT3), and expression of STAT3 target genes, Bcl-XL and cyclin D1. Furthermore, honokiol enhanced the growth inhibitory and anti-invasion activity of the EGFR-targeting agent erlotinib. Although HNSCC xenograft models did not show significant inhibition of in vivo tumor growth with honokiol treatment alone, the combination of honokiol plus cetuximab, a Food and Drug Administration–approved EGFR inhibitor for this malignancy, significantly enhanced growth inhibition. Finally, HNSCC cells rendered resistant to erlotinib retained sensitivity to the growth inhibitory effects of honokiol. Conclusions: These results suggest that honokiol may be an effective therapeutic agent in HNSCC, in which it can augment the effects of EGFR inhibitors and overcome drug resistance. Clin Cancer Res; 16(9); 2571–9. ©2010 AACR.

Kinin B2 Receptor Mediates Induction of Cyclooxygenase-2 and Is Overexpressed in Head and Neck Squamous Cell Carcinomas

Bradykinin has been shown to promote growth and migration of head and neck squamous cell carcinoma (HNSCC) cells via epidermal growth factor receptor (EGFR) transactivation. It has also been reported that bradykinin can cause the induction of cyclooxygenase-2 (COX-2), a protumorigenic enzyme, via the mitogen-activated protein kinase (MAPK) pathway in human airway cells. To determine whether COX-2 is up-regulated by bradykinin in HNSCC, the current study investigated bradykinin-induced EGFR transactivation, MAPK activation, and COX-2 expression in human HNSCC cells. Bradykinin induced a concentration- and time-dependent induction of COX-2 protein in HNSCC, which was preceded by phosphorylation of EGFR and MAPK. These effects were abolished by the B2 receptor (B2R) antagonist HOE140 but not by the B1 receptor (B1R) antagonist Lys-[Leu8]des-Arg9-bradykinin. COX-2 induction was accompanied by increased release of prostaglandin E2. No effect of a B1R agonist (des-Arg9-bradykinin) on p-MAPK or COX-2 expression was observed. B2R protein was found to be expressed in all four head and neck cell lines tested. Immunohistochemical analysis and immunoblot analysis revealed that B2R, but not B1R, was significantly overexpressed in HNSCC tumors compared with levels in normal mucosa from the same patient. In HNSCC cells, the bradykinin-induced expression of COX-2 was inhibited by the EGFR kinase inhibitor gefitinib or mitogen-activated protein kinase kinase inhibitors (PD98059 or U0126). These results suggest that EGFR and MAPK are required for COX-2 induction by bradykinin. Up-regulation of the B2R in head and neck cancers suggests that this pathway is involved in HNSCC tumorigenesis. (Mol Cancer Res 2008;6(12):1946–56)

Treatment of Triple-Negative Breast Cancer with TORC1/2 Inhibitors Sustains a Drug-Resistant and Notch-Dependent Cancer Stem Cell Population.

Approximately 30% of triple-negative breast cancers (TNBC) harbor molecular alterations in PI3K/mTOR signaling, but therapeutic inhibition of this pathway has not been effective. We hypothesized that intrinsic resistance to TORC1/2 inhibition is driven by cancer stem cell (CSC)-like populations that could be targeted to enhance the antitumor action of these drugs. Therefore, we investigated the molecular mechanisms by which PI3K/mTOR inhibitors affect the stem-like properties of TNBC cells. Treatment of established TNBC cell lines with a PI3K/mTOR inhibitor or a TORC1/2 inhibitor increased the expression of CSC markers and mammosphere formation. A CSC-specific PCR array revealed that inhibition of TORC1/2 increased FGF1 and Notch1 expression. Notch1 activity was also induced in TNBC cells treated with TORC1/2 inhibitors and associated with increased mitochondrial metabolism and FGFR1 signaling. Notably, genetic and pharmacologic blockade of Notch1 abrogated the increase in CSC markers, mammosphere formation, and in vivo tumor-initiating capacity induced by TORC1/2 inhibition. These results suggest that targeting the FGFR-mitochondrial metabolism-Notch1 axis prevents resistance to TORC1/2 inhibitors by eradicating drug-resistant CSCs in TNBC, and may thus represent an attractive therapeutic strategy to improve drug responsiveness and efficacy.

Kinome wide functional screen identifies role of Polo-like kinase 1 (PLK1) in hormone-independent, ER-positive breast cancer

Estrogen receptor (ER) α-positive breast cancers initially respond to antiestrogens but eventually become estrogen independent and recur. ER(+) breast cancer cells resistant to long-term estrogen deprivation (LTED) exhibit hormone-independent ER transcriptional activity and growth. A kinome-wide siRNA screen using a library targeting 720 kinases identified Polo-like kinase 1 (PLK1) as one of the top genes whose downregulation resulted in inhibition of estrogen-independent ER transcriptional activity and growth of LTED cells. High PLK1 mRNA and protein correlated with a high Ki-67 score in primary ER(+) breast cancers after treatment with the aromatase inhibitor letrozole. RNAi-mediated knockdown of PLK1 inhibited ER expression, estrogen-independent growth, and ER transcription in MCF7 and HCC1428 LTED cells. Pharmacologic inhibition of PLK1 with volasertib, a small-molecule ATP-competitive PLK1 inhibitor, decreased LTED cell growth, ER transcriptional activity, and ER expression. Volasertib in combination with the ER antagonist, fulvestrant, decreased MCF7 xenograft growth in ovariectomized mice more potently than each drug alone. JUNB, a component of the AP-1 complex, was expressed 16-fold higher in MCF7/LTED compared with parental MCF7 cells. Furthermore, JUNB and BCL2L1 (which encodes antiapoptotic BCL-xL) mRNA levels were markedly reduced upon volasertib treatment in MCF7/LTED cells, while they were increased in parental MCF7 cells. Finally, JUNB knockdown decreased ER expression and transcriptional activity in MCF7/LTED cells, suggesting that PLK1 drives ER expression and estrogen-independent growth via JUNB. These data support a critical role of PLK1 in acquired hormone-independent growth of ER(+) human breast cancer and is therefore a promising target in tumors that have escaped estrogen deprivation therapy.