HER3 functions as an effective therapeutic target in triple negative breast cancer to potentiate the antitumor activity of gefitinib and paclitaxel.

BACKGROUND: Triple negative breast cancer (TNBC) represents a significant clinical challenge. Chemotherapy remains the mainstay for a large part of TNBC patients, whereas drug resistance and tumor recurrence frequently occur. It is in urgent need to identify novel molecular targets for TNBC and develop effective therapy against the aggressive disease. METHODS: Immunohistochemistry was performed to examine the expression of HER3 in TNBC samples. Western blots were used to assess protein expression and activation. Cell proliferation and viability were determined by cell growth (MTS) assays. TCGA databases were analyzed to correlate HER3 mRNA expression with the clinical outcomes of TNBC patients. Specific shRNA was used to knockdown HER3 expression. IncuCyte system was utilized to monitor cell growth and migration. LIVE/DEAD Cell Imaging was to detect live and dead cells. HER3 recognition by our anti-HER3 monoclonal antibody (mAb) 4A7 was verified by ELISA, flow cytometry, and co-immunoprecipitation assays. Orthotopic tumor models were established in nude mice to determine the capability of TNBC cells forming tumors and to test if our mAb 4A7 could potentiate the antitumor activity of paclitaxel in vivo. RESULTS: Elevated expression of HER3 was observed in approximately half of the TNBC specimens and cell lines tested. Analyses of TCGA databases found that the TNBC patients with high HER3 mRNA expression in the tumors showed significantly worse overall survival (OS) and relapse-free survival (RFS) than those with low HER3 expression. Specific knockdown of HER3 markedly inhibited TNBC cell proliferation and mammosphere formation in vitro and tumor growth in vivo. Our mAb 4A7 abrogated heregulin (a ligand for HER3), but not SDF-1 (a ligand for CXCR4)-induced enhancement of TNBC cell migration. Combinations of 4A7 and the EGFR-tyrosine kinase inhibitor (TKI) gefitinib dramatically decreased the levels of phosphorylated HER3, EGFR, Akt, and ERK1/2 in TNBC cells and potently induced growth inhibition and cell death. Moreover, 4A7 in combination with paclitaxel exerted significant antitumor activity against TNBC in vitro and in vivo. CONCLUSIONS: Our data demonstrate that increased HER3 is an effective therapeutic target for TNBC and our anti-HER3 mAb (4A7) may enhance the efficacy of gefitinib or paclitaxel in TNBC.

HER3 targeting augments the efficacy of panobinostat in claudin-low triple-negative breast cancer cells.

Patients with triple-negative breast cancer (TNBC) have a poor prognosis and high relapse rate due to limited therapeutic options. This study was conducted to determine the mechanisms of action of panobinostat, a pan-inhibitor of histone deacetylase (HDAC) and FDA-approved medication for multiple myeloma, in TNBC and to provide a rationale for effective drug combinations against this aggressive disease. RNA sequencing analyses of the claudin-low (CL) TNBC (MDA-MB-231) cells untreated or treated with panobinostat were performed to identify the differentially expressed genes. Adaptive alterations in gene expression were analyzed and validated in additional CL TNBC cells. Tumor xenograft models were used to test the in vivo antitumor activity of panobinostat alone or its combinations with gefitinib, an EGFR-tyrosine kinase inhibitor (TKI). Panobinostat potently inhibited proliferation and induced apoptosis in all TNBC cells tested. However, in CL TNBC cells, this HDAC inhibitor markedly enhanced expression of HER3, which interacted with EGFR to activate both receptors and Akt signaling pathways. Combinations of panobinostat and gefitinib synergistically suppressed CL TNBC cell proliferation and promoted apoptosis in vitro and in vivo. Upregulation of HER3 compromises the efficacy of panobinostat in CL TNBC. Inactivation of HER3 combined with panobinostat represents a practical approach to combat CL TNBC.

Trastuzumab-resistant breast cancer cells-derived tumor xenograft models exhibit distinct sensitivity to lapatinib treatment in vivo.

BACKGROUND: Resistance to HER2-targeted therapies, including the monoclonal antibody trastuzumab and tyrosine kinase inhibitor lapatinib, frequently occurs and currently represents a significant clinical challenge in the management of HER2-positive breast cancer. We previously showed that the trastuzumab-resistant SKBR3-pool2 and BT474-HR20 sublines were refractory to lapatinib in vitro as compared to the parental SKBR3 and BT474 cells, respectively. The in vivo efficacy of lapatinib against trastuzumab-resistant breast cancer remained unclear. RESULTS: In tumor xenograft models, both SKBR3-pool2- and BT474-HR20-derived tumors retained their resistance phenotype to trastuzumab; however, those tumors responded differently to the treatment with lapatinib. While lapatinib markedly suppressed growth of SKBR3-pool2-derived tumors, it slightly attenuated BT474-HR20 tumor growth. Immunohistochemistry analyses revealed that lapatinib neither affected the expression of HER3, nor altered the levels of phosphorylated HER3 and FOXO3a in vivo. Interestingly, lapatinib treatment significantly increased the levels of phosphorylated Akt and upregulated the expression of insulin receptor substrate-1 (IRS1) in the tumors-derived from BT474-HR20, but not SKBR3-pool2 cells. CONCLUSIONS: Our data indicated that SKBR3-pool2-derived tumors were highly sensitive to lapatinib treatment, whereas BT474-HR20 tumors exhibited resistance to lapatinib. It seemed that the inefficacy of lapatinib against BT474-HR20 tumors in vivo was attributed to lapatinib-induced upregulation of IRS1 and activation of Akt. Thus, the tumor xenograft models-derived from SKBR3-pool2 and BT474-HR20 cells serve as an excellent in vivo system to test the efficacy of other HER2-targeted therapies and novel agents to overcome trastuzumab resistance against HER2-positive breast cancer.

ALKBH5-Mediated m6A Demethylation of GLUT4 mRNA Promotes Glycolysis and Resistance to HER2-Targeted Therapy in Breast Cancer.

Resistance to HER2-targeted therapy represents a significant challenge for the successful treatment of patients with breast cancer with HER2-positive tumors. Through a global mass spectrometry-based proteomics approach, we discovered that the expression of the N6-methyladenosine (m6A) demethylase ALKBH5 was significantly upregulated in HER2-targeted therapy-resistant breast cancer cells. Elevated expression of ALKBH5 was sufficient to confer resistance to HER2-targeted therapy, and specific knockdown of ALKBH5 rescued the efficacy of trastuzumab and lapatinib in resistant breast cancer cells. Mechanistically, ALKBH5 promoted m6A demethylation of GLUT4 mRNA and increased GLUT4 mRNA stability in a YTHDF2-dependent manner, resulting in enhanced glycolysis in resistant breast cancer cells. In breast cancer tissues obtained from patients with poor response to HER2-targeted therapy, increased expression of ALKBH5 or GLUT4 was observed and was significantly associated with poor prognosis in the patients. Moreover, suppression of GLUT4 via genetic knockdown or pharmacologic targeting with a specific inhibitor profoundly restored the response of resistant breast cancer cells to trastuzumab and lapatinib, both in vitro and in vivo. In conclusion, ALKBH5-mediated m6A demethylation of GLUT4 mRNA promotes resistance to HER2-targeted therapy, and targeting the ALKBH5/GLUT4 axis has therapeutic potential for treating patients with breast cancer refractory to HER2-targeted therapies. SIGNIFICANCE: GLUT4 upregulation by ALKBH5-mediated m6A demethylation induces glycolysis and resistance to HER2-targeted therapy and represents a potential therapeutic target for treating HER2-positive breast cancer.

Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows promising antitumor activity in preclinical studies. However, the efficacy of recombinant TRAIL in clinical trials is compromised by its short serum half-life and low in vivo stability. Induction of endogenous TRAIL may overcome the limitations and become a new strategy for cancer treatment. Here, we discovered that metformin increased TRAIL expression and induced apoptosis in triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) cells. Metformin did not alter the expression of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Metformin-upregulated TRAIL was secreted into conditioned medium (CM) and found to be functional, since the CM promoted TNBC cells undergoing apoptosis, which was abrogated by a recombinant TRAIL-R2-Fc chimera. Moreover, blockade of TRAIL binding to DR4/DR5 or specific knockdown of TRAIL expression significantly attenuated metformin-induced apoptosis. Studies with a tumor xenograft model revealed that metformin not only significantly inhibited tumor growth but also elicited apoptosis and enhanced TRAIL expression in vivo. Collectively, we have demonstrated that upregulation of TRAIL and activation of death receptor signaling are pivotal for metformin-induced apoptosis in TNBC and NSCLC cells. Our studies identify a novel mechanism of action of metformin exhibiting potent antitumor activity via induction of endogenous TRAIL.

Alcohol consumption increases susceptibility to pneumococcal pneumonia in a humanized murine HIV model mediated by intestinal dysbiosis.

Alcohol use in persons living with HIV (PLWH) worsens the severity of bacterial pneumonia. However, the exact mechanism(s) by which this occurs remain ill-defined. We hypothesized that alcohol in the setting of HIV infection decreases Streptococcus pneumoniae clearance from the lung through mechanisms mediated by the gut microbiota. Humanized BLT (bone marrow, liver, thymus) mice were infected with 1 × 104 TCID50 of HIV (BAL and JRCSF strains) via intraperitoneal (i.p.) injection. One week post-HIV infection, animals were switched to a Lieber-DeCarli 5% ethanol diet or an isocaloric control diet for 10 days. Alcohol-fed animals were also given two binges of 2 g/kg ethanol on days 5 and 10. Feces were also collected, banked, and the community structures were analyzed. Mice were then infected with 1 × 105 CFU (colony-forming units) of S. pneumoniae and were sacrificed 48 h later. HIV-infected mice had viral loads of ∼2 × 104 copies/mL of blood 1 week post-infection, and exhibited an ∼57% decrease in the number of circulating CD4+ T cells at the time of sacrifice. Fecal microbial community structure was significantly different in each of the feeding groups, as well as with HIV infection. Alcohol-fed mice had a significantly higher burden of S. pneumoniae 48 h post-infection, regardless of HIV status. In follow-up experiments, female C57BL/6 mice were treated with a cocktail of antibiotics daily for 2 weeks and recolonized by gavage with intestinal microbiota from HIV+ ethanol-fed, HIV+ pair-fed, HIV- ethanol-fed, or HIV- pair-fed mice. Recolonized mice were then infected with S. pneumoniae and were sacrificed 48 h later. The intestinal microbiota from alcohol-fed mice (regardless of HIV status) significantly impaired clearance of S. pneumoniae. Collectively, these data indicate that alcohol feeding, as well as alcohol-associated intestinal dysbiosis, compromise pulmonary host defenses against pneumococcal pneumonia. Determining whether HIV infection acts synergistically with alcohol use in impairing pulmonary host defenses will require additional study.

B cell and antibody responses in mice induced by a putative cell surface peptidase of Pneumocystis murina protect against experimental infection.

RATIONALE: Pneumocystis pneumonia is a major cause of morbidity and mortality in HIV-infected subjects, cancer patients undergoing chemotherapy and solid organ transplant recipients. No vaccine is currently available. By chemical labeling coupled with proteomic approach, we have identified a putative surface protein (SPD1, Broad Institute gene accession number PNEG_01848) derived from single suspended P. murina cysts. SPD1 was expressed in an insect cell line and tested for vaccine development. METHODS: Mice were immunized with SPD1 plus adjuvant MF-59 by subcutaneous injection. Three weeks after the last immunization, CD4+ cells were depleted with anti-CD4 antibody GK1.5. The mice were then challenged with 2×105Pneumocystis organisms. Mice were sacrificed at 4 and 6weeks after PC challenge. Spleen/lung cells and serum were harvested. B cells and memory B cells were assessed via flow cytometry. Specific Pneumocystis IgG antibody was measured by ELISA before and after challenge. Infection burden was measured as real-time PCR for P. murina rRNA. RESULTS: Normal mice infected with Pneumocystis mounted a serum IgG antibody response to SPD1. Serum from rhesus macaques exposed to Pneumocystis showed a similar serum IgG response to purified SPD1. SPD1 immunization increased B cell and memory B cell absolute cell counts in CD4-depleted Balb/c mice post Pneumocystis challenge in spleen and lung. Immunization with SPD1 significantly increased specific Pneumocystis IgG antibody production before and after challenge. Mice immunized with SPD1 showed significantly decreased P. murina copy number compared with mice that did not receive SPD1 at 6weeks after challenge. CONCLUSION: Immunization with SPD1 provides protective efficacy against P. murina infection. SPD1 protection against Pneumocystis challenge is associated with enhanced memory B cell production and higher anti-Pneumocystis IgG antibody production. SPD1 is a potential vaccine candidate to prevent or treat pulmonary infection with Pneumocystis.

Oral Immunization of Mice with Live Pneumocystis murina Protects against Pneumocystis Pneumonia.

Pneumocystis pneumonia is a major cause of morbidity and mortality in immunocompromised patients, particularly those infected with HIV. In this study, we evaluated the potential of oral immunization with live Pneumocystis to elicit protection against respiratory infection with Pneumocystis murina. C57BL/6 mice vaccinated with live P. murina using a prime-boost vaccination strategy were protected from a subsequent lung challenge with P. murina at 2, 7, 14, and 28 d postinfection even after CD4(+) T cell depletion. Specifically, vaccinated immunocompetent mice had significantly faster clearance than unvaccinated immunocompetent mice and unvaccinated CD4-depleted mice remained persistently infected with P. murina. Vaccination also increased numbers of CD4(+) T cells, CD8(+) T cells, CD19(+) B cells, and CD11b(+) macrophages in the lungs following respiratory infection. In addition, levels of lung, serum, and fecal P. murina-specific IgG and IgA were increased in vaccinated animals. Furthermore, administration of serum from vaccinated mice significantly reduced Pneumocystis lung burden in infected animals compared with control serum. We also found that the diversity of the intestinal microbial community was altered by oral immunization with P. murina. To our knowledge, our data demonstrate for the first time that an oral vaccination strategy prevents Pneumocystis infection.

Suppression of the macrophage proteasome by ethanol impairs MHC class I antigen processing and presentation.

Alcohol binge-drinking (acute ethanol consumption) is immunosuppressive and alters both the innate and adaptive arms of the immune system. Antigen presentation by macrophages (and other antigen presenting cells) represents an important function of the innate immune system that, in part, determines the outcome of the host immune response. Ethanol has been shown to suppress antigen presentation in antigen presenting cells though mechanisms of this impairment are not well understood. The constitutive and immunoproteasomes are important components of the cellular proteolytic machinery responsible for the initial steps critical to the generation of MHC Class I peptides for antigen presentation. In this study, we used an in-vitro cell culture model of acute alcohol exposure to study the effect of ethanol on the proteasome function in RAW 264.7 cells. Additionally, primary murine peritoneal macrophages obtained by peritoneal lavage from C57BL/6 mice were used to confirm our cell culture findings. We demonstrate that ethanol impairs proteasome function in peritoneal macrophages through suppression of chymotrypsin-like (Cht-L) proteasome activity as well as composition of the immunoproteasome subunit LMP7. Using primary murine peritoneal macrophages, we have further demonstrated that, ethanol-induced impairment of the proteasome function suppresses processing of antigenic proteins and peptides by the macrophage and in turn suppresses the presentation of these antigens to cells of adaptive immunity. The results of this study provide an important mechanism to explain the immunosuppressive effects of acute ethanol exposure.

Lymphocyte apoptosis in murine Pneumocystis pneumonia.

BACKGROUND: Apoptosis of lymphocytes is important in the termination of an immune response to infection but has also been shown to have detrimental effects in animal models of systemic infection and sepsis. We sought to characterize lymphocyte apoptosis in an animal model of pneumonia due to Pneumocystis murina, an infection localized to the lungs. METHODS: Control mice and mice depleted of CD4+ lymphocytes were inoculated with Pneumocystis. Apoptosis of lung and spleen lymphocytes was assayed by flow cytometry and PCR assay of apoptotic proteins. RESULTS: In control mice, apoptosis of lung lymphocytes was maximal just after the infection was cleared from lung tissue and then declined. However, in CD4-depleted mice, apoptosis was also upregulated in recruited lymphocytes in spite of progressive infection. In splenic lymphocytes, apoptosis was observed early at 1 week after inoculation and then declined. Apoptosis of lung lymphocytes in control mice was associated with a decrease in mRNA for Bcl-2 and an increase in mRNA for Bim. In CD4-depleted mice, lavaged CD8+ cells did change intracellular Bcl-2 but showed increased mRNA for Bim. CONCLUSION: Apoptosis of both pulmonary and extrapulmonary lymphocytes is part of the normal host response to Pneumocystis but is also triggered in CD4-deficient animals with progressive infection. In normal mice apoptosis of pulmonary lymphocytes may serve to terminate the immune response in lung tissue. Apoptosis of lung lymphocytes takes place via both the intrinsic and extrinsic apoptotic pathways and is associated with changes in both pro- and anti-apoptotic proteins.

Interleukin-12 and host defense against murine Pneumocystis pneumonia.

Little is known about the role of the cytokine interleukin-12 (IL-12) in Pneumocystis pneumonia or its potential use as immunotherapy. We asked whether release of IL-12 is part of the normal host response to this infection and whether local treatment with IL-12 or gene transfer of IL-12 could accelerate clearance of infection. IL-12 was assayed by enzyme-linked immunosorbent assay in normal mice and in mice deficient in IL-12 after inoculation of Pneumocystis carinii. P. carinii-infected mice were treated with local instillation of IL-12 and gene transfer of the IL-12 gene. Inoculation of P. carinii into normal mice evoked a brisk release of IL-12 into lung tissue, and IL-12 P35-deficient mice showed delayed clearance of infection measured by PCR for P. carinii rRNA. In control mice, intranasal recombinant IL-12 accelerated clearance of infection, and this was associated with increased recruitment of inflammatory cells into lavage fluid and increased release of tumor necrosis factor alpha, IL-12, and gamma interferon. Similar results were observed in infected mice depleted of CD4+ lymphocytes by using in vivo transfer of the IL-12 gene in a replication-deficient adenoviral vector. IL-12 is part of the normal host response to infection with P. carinii. IL-12 therapy can enhance host resistance to infection in both normal mice and mice depleted of CD4+ T lymphocytes. A treatment effect of IL-12 is mediated through enhanced inflammatory cell recruitment into lung tissue and increased tissue concentrations of proinflammatory cytokines.

CXCR3 and IFN protein-10 in Pneumocystis pneumonia.

We have previously shown that Tc1 CD8(+) T cells have in vitro and in vivo effector activity against Pneumocystis (PC) infection in mice. Because these cells have preferential expression of CXCR3, we investigated whether CXCR3 was required for host defense activity against PC. Mice deficient in CXCR3 but CD4(+) T cell intact, showed an initial delay but were able to clear the infectious challenge, indicating that CXCR3 signaling is not essential for clearance of PC. CD4-depleted mice had lower levels of monokine induced by IFN-gamma, IFN protein-10 (IP-10), and IFN-inducible T cell alpha-chemoattractant at day 7 of infection and are permissive to PC infection. Overexpression of IP-10 in the lungs by adenoviral gene transfer did not accelerate clearance of infection in control mice but accelerated clearance by day 28 in mice depleted of CD4(+) T cells. This effect was associated with increased recruitment of CD8(+) T to the lungs with higher CXCR3(+) expression levels and enhanced IFN-gamma secretion upon in vitro activation compared with control mice. These results indicate that the CXCR3 chemokines are part of the host defense response to PC, and that IP-10 can direct Tc1 CD8(+) T cell recruitment to the lungs and contribute to host defense against PC even in the absence of CD4(+) T cells.

Conditional expression of interferon-gamma to enhance host responses to pulmonary bacterial infection.

Strategies to augment host defense against pulmonary infection run the risk of inducing excess pulmonary inflammation and tissue injury. To address this problem, we investigated conditional expression in lung tissue of the murine interferon-gamma (IFN-gamma) transgene. A recombinant adenoviral vector (AdTetIFN) was constructed by placing a murine IFN-gamma cDNA downstream of a tetracycline (Tet)-responsive promoter, inserted into a replication-defective adenoviral vector. Co-infection of target cells with AdTetIFN and a second vector encoding a reverse tetracycline controlled transactivator allowed doxycycline (Dox)-regulated IFN-gamma production. We then administered 10(8) plaque-forming units (PFU) of AdTetIFN to mice by intratracheal injection. When the mice were provided with Dox in drinking water (0.5mg/ml in 5% sucrose), there was significant release of IFN-gamma in lavage fluid by ELISA in comparison to mice on water/sucrose alone (399+/-74 pg/ml vs undetectable, p<0.01). IFN-gamma in lavage fluid was associated with upregulation of Class II Major histocompatibility complex markers on alveolar macrophages by flow cytometry, suggesting macrophage activation. We then injected AdTetIFN into mice three days prior to challenge with 10(4) CFU Klebsiella pneumoniae. Test mice were maintained on water+Dox and control mice on water+sucrose. Bacterial burden was assayed in lung tissue at serial intervals. At 24h after challenge, mice on doxycycline had significantly lower infection burden in comparison to mice on water/sucrose (0.77+/-0.05 colony forming units/lung for 10(8) PFU AdTetIFN plus Dox compared to 1.4+/-0.11 colony-forming units/lung for AdTetIFN without Dox, p<0.05). Survival of the vector treated mice given doxycycline in drinking water was also enhanced. Microscopic examination of lavaged cells showed a significant increase in pulmonary neutrophils in the AdTetIFN+Dox mice in comparison to AdTetIFN+sucrose mice (16+/-1.0 x 10(5) vs 10+0.8 cells/lung, p<0.05). We conclude that local release of IFN-gamma can be selectively activated to enhance neutrophil recruitment and host resistance to bacterial pneumonia.

Local delivery of the viral interleukin-10 gene suppresses tissue inflammation in murine Pneumocystis carinii infection.

The relationship between tissue inflammation and clearance of the opportunistic pathogen Pneumocystis carinii is poorly understood. We asked whether the anti-inflammatory cytokine interleukin-10 (IL-10) is released during the host response to infection with P. carinii and whether local delivery of the IL-10 gene could suppress tissue inflammatory responses without compromising clearance of infection. Control and CD4-depleted mice were inoculated with P. carinii, and at serial intervals after inoculation, lung tissue was assayed for IL-10 by enzyme-linked immunosorbent assay. We found that IL-10 was released in lung tissue in control mice and was present in higher concentrations in CD4-depleted mice with progressive infection. Control and CD4-depleted mice were then pretreated with 10(9) PFU of intratracheally administered adenoviral vector containing the viral IL-10 gene or the luciferase gene followed by inoculation with P. carinii. Pretreatment with viral IL-10 did not alter clearance of infection in control mice or severity of infection in CD4-depleted mice but did decrease tissue inflammation. We then asked whether gene transfer of viral IL-10 could decrease tissue inflammation during immune reconstitution. In these experiments, immunodeficient scid mice were inoculated with P. carinii and were heavily infected after 4 weeks. When these mice are immunologically reconstituted by intravenous administration of spleen cells from normal mice, a hyperinflammatory reaction developed in lung tissue, associated with high mortality. In comparison to control mice, mice treated with viral IL-10 prior to reconstitution showed significantly decreased lung wet weight, bronchoalveolar lavage fluid (BALF) lactate dehydrogenase, and BALF neutrophils. In contrast, infection intensity, as measured by PCR for P. carinii rRNA, was unchanged between the IL-10 and luciferase groups. Survival was also improved in the IL-10-treated mice. We conclude that release of IL-10 is part of the host response to infection with P. carinii and that gene therapy with viral IL-10 can lessen excessive tissue inflammation without altering pathogen clearance. In the setting of immune reconstitution and P. carinii pneumonia, pretreatment with the viral IL-10 gene decreases excessive tissue inflammation and improves survival. These results are relevant to acute respiratory failure after initiation of antibiotic treatment for human P. carinii pneumonia and to immune reconstitution syndromes in human immunodeficiency virus-positive patients started on highly active antiretroviral therapy.