Vitamin A and D3 combinations reduce breast cancer tumor load in a postmenopausal MCF-7 xenograft mouse model in a dose- and time- dependent manner

Manuka honey (MH) exhibits potential antitumor activity in preclinical models of a number of human cancers. Treatment in vitro with MH at concentrations ranging from 0.3 to 5.0% (w/v) led to significant dose-dependent inhibition of proliferation of human breast cancer MCF-7 cells, but anti-proliferative effects of MH were less pronounced in MDA-MB-231 breast cancer cells. Effects of MH were also tested on non-malignant human mammary epithelial cells (HMECs) at 2.5% w/v, and it was found that MH reduced the proliferation of MCF-7 cells but not that of HMECs. Notably, the antitumor activity of MH was in the range of that exerted by treatment of MCF-7 cells with the antiestrogen tamoxifen. Further, MH treatment stimulated apoptosis of MCF-7 cells in vitro, with most cells exhibiting acute and significant levels of apoptosis that correlated with PARP activation. Additionally, the effects of MH induced the activation of AMPK and inhibition of AKT/mTOR downstream signaling. Treatment of MCF7 cells with increased concentrations of MH induced AMPK phosphorylation in a dose-dependent manner that was accompanied by inhibition of phosphorylation of AKT and mTOR downstream effector protein S6. In addition, MH reduced phosphorylated STAT3 levels in vitro, which may correlate with MH and AMPK-mediated anti-inflammatory properties. Further, in vivo, MH administered alone significantly inhibited the growth of established MCF-7 tumors in nude mice by 84%, resulting in an observable reduction in tumor volume. Our findings highlight the need for further research into the use of natural compounds, such as MH, for antitumor efficacy and potential chemoprevention and investigation of molecular pathways underlying these actions.

Human type I fatty acid synthase has been proposed as a chemotherapeutic target for the treatment of breast cancer based on the inactivation of human beta-ketoacyl synthase activity by cerulenin. Triclosan, a common antibiotic, functions by inhibiting the enoyl-reductase enzymes of type II fatty acid synthases in susceptible bacteria. If triclosan is an inhibitor of human fatty acid synthase and if inhibition of fatty acid synthase is toxic to breast cancer cell lines, triclosan could prove to be a lead compound for the treatment of breast cancer. Consequently, the inhibitory activity of triclosan against vertebrate type I fatty acid synthases and its effects on breast cancer lines in cell culture were investigated. The inhibitory activities of triclosan against human and goose fatty acid synthases and each of the partial reactions were investigated using spectrophotometric assays. The ability of triclosan at various concentrations to inhibit growth and reduce the viability of MCF-7 and SKBr-3 cells in culture was evaluated. Kinetic studies showed triclosan to be a slow binding inhibitor of human and goose type I fatty acid synthase and to inhibit the partial activity of enoyl-reductase with IC(50) values between 10 and 50 microM. Triclosan at similar concentrations was also shown to inhibit both viability and growth of MCF-7 and SKBr-3 cells in culture. The results corroborate the hypothesis that fatty acid synthase may be a target of breast cancer chemotherapy and suggest that inhibitors of the enoyl-reductase partial activity of fatty acid synthase may have chemotherapeutic potential.

Cytochrome P450 3A4 (CYP3A4) promotes ER+HER2- breast cancer cell proliferation and survival, in part, by biosynthesis of epoxyeicosatrienoic acids (EETs). EETs are known to regulate mitochondrial function in non-transformed cells, but the roles of CYP3A4 and EETs in regulation of breast cancer bioenergetics are unknown. Hexyl-benzyl-biguanide (HBB) is useful probe of CYP3A4 epoxygenase activity and selectively inhibits EET biosynthesis (IC50 = 9 uM vs. IC50 = 50 uM for CYP2C8). HBB caused depolarization of mitochondria in MCF-7 cells, while (±)-14,15-EET provided partial protection. The soluble epoxide hydrolase (sEH) inhibitor t-AUCB ameliorated inhibition of oxygen consumption rates (OCR) by HBB (20 uM), while there was no effect on extracellular acidification rate (ECAR), indicating that the primary effect of HBB is on OCR. At 30 minutes, HBB added to MCF-7 cells transiently suppressed phosphorylation of pyruvate kinase muscle isozyme 2 (PKM2) on Tyr-105, which has been reported to favor enzymatically inactive dimer over active tetramer. Suppression of phosphorylated PKM2 correlated with subsequent PKM2 tetramer formation and increase of intracellular pyruvate and extracellular lactate at 1 hour. The (±)-14,15-EET regioisomer reduced the pro-glycolytic PKM2 tetramer at 1 hour, suggesting that HBB may promote PKM2 tetramer, in part, through reduction of EET. Prolonged exposure to HBB (20 uM) in cultured cells activated phosphorylation of PKM2 on Tyr-105, but there was increased cellular necrosis correlating with reduced mitochondrial respiration and reduction of ATP stores, indicating that loss of respiration was the dominant effect. HBB inhibited the ER+HER2- MCF-7 xenograft, similar to CYP3A4 silencing. HBB promoted phosphorylation of intratumoral PKM2 on Tyr-105, consistent with long-term exposure to HBB in cultured MCF-7 cells. Notably, MCF-7 tumor response to HBB did not correlate with phosphorylation of AMPK-alpha on Thr-172, a marker of AMPK activation. Metformin (5 mM) exhibited no effect on PKM2 or its phosphorylation in cultured MCF-7 cells. Together, these results indicate that part of the inhibitory effect of HBB on ER+HER2- breast cancer is mediated through inhibition of respiration. Significance: These results establish HBB as a useful chemical probe of respiration, with indirect effects on PKM2 regulation. HBB may also be useful as a potential therapeutic candidate for ER+HER2- breast cancer. Citation Format: Zhijun Guo, Irina Sevrioukova, Eric Hanse, Xia Zhang, Ilia Denisov, Ting-Lan Chiu, Rebecca Cuellar, Christian Torres, Julia Wulfkuhle, Emanuel Petricoin, Qing Cao, Haitao Chu, Beverly Norris, Robert Schumacher, Ameeta Kelekar, Ian Blair, Jorge Capdevila, John Falck, Thomas Poulos, Steven Sligar, Gunda Georg, Elizabeth Amin, David A. Potter. Hexyl-benzyl-biguanide (HBB) potently and selectively inhibits CYP3A4 epoxygenase activity and inhibits EET stabilization of mitochondrial respiration in ER+HER2- breast cancer cells, inducing glycolysis and pyruvate biosynthesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 44.

As an antineoplastic antibiotic, doxorubicin (DOX) effectively inhibits RNA and DNA synthesis. However, its application is limited by side effects and drug resistance. This study established a xenograft mouse model of breast cancer (BC) to investigate the therapeutic efficacy of a gene/drug nano-delivery system combining vascular endothelial growth factor (VEGF) silencing with DOX for treating BC. Poly (lactic acid) (PLA) was utilized as the carrier material to prepare nanoparticles (NPs) loaded with VEGF interference RNA (siRNA) and the chemotherapeutic drug DOX (PLA/DOX-NPs and PLA/DOX-siVEGF-NPs). The characterization and drug release of NPs were analyzed. NPs’ cytotoxicity was determined by CCK-8 assay with HUVEC and MCF-7 cells. The BC xenograft (BCX) model was established by injecting MCF-7 cells into the mammary fat pads of nude mice. The differences in tumor weight and tumor inhibition rate were analyzed after treatment with DOX alone, PLA/DOX-NPs, and PLA/DOX-siVEGF-NPs. Immunohistochemical staining was employed to examine Ki-67 positive expression rate in the BCXs. Western blot was used to detect Ki-67 and VEGF protein expression levels in transplanted tumor tissues. Additionally, a suspension of BCX cells was injected subcutaneously into healthy nude mice to assess tumor growth after secondary engraftment. Both PLA/DOX-NPs and PLA/DOX-siVEGF-NPs demonstrated sustained release of DOX in buffer media at varying pH values, with no visible difference in inhibiting human umbilical vein endothelial cells (HUVECs) proliferation. After establishment of the BCX mouse model, compared to DOX group, the PLA/DOX-NPs group and PLA/DOX-siVEGF-NPs group exhibited a significant reduction in relative tumor volume and Ki-67 index, along with an increased tumor inhibition rate. Furthermore, after secondary tumor formation, both tumor volume and size were markedly reduced (P<0.05). There were statistically significant differences in various parameters between the PLA/DOX-NPs group and the PLA/DOX-siVEGF-NPs group (P<0.05). The prepared PLA/DOX-siVEGF-NPs demonstrated low toxicity to normal cells and strongly inhibited the proliferation of MCF-7 BC cells in vitro. Moreover, PLA/DOX-siVEGF-NPs effectively inhibited the growth of BCXs in nude mice and suppressed Ki-67 positive expression. This treatment also reduced the malignant differentiation of the tumors and inhibited tumor recurrence. Key words: xenograft mouse model, doxorubicin; nanoparticles; breast cancer; vascular endothelial growth factor.

Background: Present line indications for pembrolizumab are for patients with metastatic or unresectable recurrent (i.e. incurable) head and neck squamous cell carcinoma (HNSCC). However, only a minority of patients on immunotherapy will realize a durable survival benefit because >80% of patients with metastatic HNSCC do not respond to PD-1 blockade. Tumor microenvironment mesenchymal stem cells (MSCs) and cancer associated fibroblasts (CAFs) have been reported significantly contribute to chemotherapy and radiation resistance. Moreover, MSCs have been shown to contribute to an immunosuppressive tumor microenvironment by upregulating PD-L1 in breast cancer models. We have previously shown crenolanib improves MSC mediated cisplatin resistance in vitro through modulation of MSC-mediated activation of AKT signaling, therefore we hypothesize that targeting MSCs may be of therapeutic benefit alone and in combination with anti-PD1 immunotherapy. Methods: Oral cancer was induced in the buccal space of C56/BL6 mice with the murine oral cancer cell lines MOC1 or PD-1 resistant cell line, MOC2. When tumors reached approximately 5 × 5 mm, mice were treated with 15 mg/kg (low dose) or 30 mg/kg crenolanib (high dose) for five consecutive days over 3 weeks. Tissue were harvested for immunohistochemistry and immunofluorescence analysis and ex vivo cell cultures prepared for analysis by western immunoblotting and flow cytometry. Results: There was a significant reduction in tumor volume in mice bearing MOC1 and MOC2 tumors (p<0.03) treated with either low or high dose crenolanib compared to vehicle control. Overall survival was also significantly improved in mice bearing MOC1 tumors treated with high dose crenolanib compared to mice treated with vehicle control (p<0.04) and approached significance in MOC2 mice treated with low dose crenolanib. Tumor sections were imaged by immunofluorescence microscopy. There was a decrease in expression of PDGFR-α on MOC1 tumor cells and α-SMA on tumor microenvironment stromal cells in mice treated with crenolanib compared to vehicle control, suggesting crenolanib targets both cell types. There was a significant reduction in tumor volume (p<0.0001) and improved overall survival (p<0.0004) in mice bearing MOC2 tumors treated with combination crenolanib plus pembrolizumab compared to vehicle plus pembrolizumab. Conclusions: Preliminary in vivo data suggests crenolanib may be efficacious when used in combination with anti-PD1 immunotherapy by inhibiting the immunosuppressive effects of tumor microenvironment MSCs. Citation Format: Xiangfeng Shen, Katherine Gonzalez, Rico Castillo, Ashlyn Rickard, Yvonne Mowery, Tammara Watts. Crenolanib improves PD-1 response and overall survival in immune checkpoint inhibitor resistant murine models of oral squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-093.

Desmopressin is a synthetic analogue of the antidiuretic hormone vasopressin. It has recently been demonstrated to inhibit tumor progression and metastasis in breast cancer models. Docetaxel is a chemotherapy agent for castrate-resistant prostate cancer (CRPC). In this study, the ability of CRPC cells to grow and develop in vivo tumors in an animal model was evaluated, in order to investigate the anti-tumor effect of desmopressin in combination with docetaxel. The CRPC cell line PC3 was used for orthotopic inoculation in male athymic nude mice. The mice were randomly assigned to one of the four treatment groups: Control, docetaxel, desmopressin or combination therapy. Following the last treatment, tumors were excised and measured. Blood samples were processed for CTC analysis. Docetaxel treatment resulted in a significant reduction in tumor volume compared to control. The combination therapy resulted in even more significant reduction (31.2%) in tumor volume. There was a complete absence of CTCs in the combination group. Our pilot study demonstrated an enhanced efficacy of docetaxel-based therapy in combination with desmopressin.

Vitamin D(3) compounds are currently in clinical trials for human breast cancer and offer an alternative approach to anti-hormonal therapies for this disease. 1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active form of vitamin D(3), induces apoptosis in breast cancer cells and tumors, but the underlying mechanisms are poorly characterized. In these studies, we focused on the role of caspase activation and mitochondrial disruption in 1alpha,25(OH)(2)D(3)-mediated apoptosis in breast cancer cells (MCF-7) in vitro. The effect of 1alpha,25(OH)(2)D(3) on MCF-7 cells was compared with that of tumor necrosis factor alpha, which induces apoptosis via a caspase-dependent pathway. Our major findings are that 1alpha,25(OH)(2)D(3) induces apoptosis in MCF-7 cells by disruption of mitochondrial function, which is associated with Bax translocation to mitochondria, cytochrome c release, and production of reactive oxygen species. Moreover, we show that Bax translocation and mitochondrial disruption do not occur after 1alpha,25(OH)(2)D(3) treatment of a MCF-7 cell clone selected for resistance to 1alpha,25(OH)(2)D(3)-mediated apoptosis. These mitochondrial effects of 1alpha,25(OH)(2)D(3) do not require caspase activation, since they are not blocked by the cell-permeable caspase inhibitor z-Val-Ala-Asp-fluoromethylketone. Although caspase inhibition blocks 1alpha,25(OH)(2)D(3)-mediated events downstream of mitochondria such as poly(ADP-ribose) polymerase cleavage, external display of phosphatidylserine, and DNA fragmentation, MCF-7 cells still execute apoptosis in the presence of z-Val-Ala-Asp-fluoromethylketone, indicating that the commitment to 1alpha,25(OH)(2)D(3)-mediated cell death is caspase-independent.

We aimed to assess the effects of a cocktail comprising three specific anti- HER2 scFvs on breast tumor formation in a xenograft mouse model and to evaluate quantitative changes in the tumor using stereological analysis. Three specific anti-HER2 phage antibodies were produced from a scFv-library using phage display technology. The cell binding capacities of the antibodies were assessed via FACS analysis. Soluble forms of the antibodies were prepared by infecting HB2151-E. coli cells and purified using a centrifugal ultrafiltration method. The purification process was evaluated by SDSPAGE analysis. Two forms of scFv cocktails were prepared, soluble scFv and phage-scFv cocktail, which contained an equal amount/phage of each of the three antibodies. Inbred female BALB/c mice were pretreated with 5 and 20 mg/kg of the soluble scFv cocktail and 1011 phage-scFv cocktail/ kg. The mice were then injected with 2×106 SKBR-3 human breast cancer cells. Total tumor, inflammatory and non-inflammatory volumes were estimated using the Cavalieri principle after preparing photomicrograph slides. The anti-HER2 scFvs showed significantly higher binding to SKBR-3 cells compared to the isotype control. SDS-PAGE analysis confirmed the high purification of the scFvs. Stereological analysis revealed that the group pretreated with 20 mg/kg of the soluble scFv cocktail exhibited the highest reductions in total tumor volume, non-inflammatory volume, and inflammatory volume, with reductions of 73%, 78%, and 72%, respectively, compared to PBS-pretreated mice (P-value < 0.0001). The volumetric ratio of necrotic tissue to total tumor volume increased by 2.2-fold and 2- fold in the 20 mg/kg of soluble scFv cocktail and phage-scFv cocktail groups, respectively, compared to the PBS-treated mice (P-value < 0.05). Pre-treatment with a 20 mg/kg anti-HER2 scFv cocktail resulted in a significant reduction in tumor volume and increased necrotic area in a human breast cancer xenograft model, indicating the remarkable anti-tumor effect of the cocktail in vivo.

BACKGROUND: Estrogen Receptor (ER) + breast cancers (BC) comprise over 70% of BC cases and can be targeted via ER modulated therapies. Despite this, ER+BC patients can experience recurrence within 20 years and the majority of BC related deaths can be attributed to metastatic ER+BC. These distant metastases commonly become diagnosed as endocrine therapy resistant. Thus, there is an unmet need to identify novel biomarkers for treating ER+ patients with metastasis. We have identified a tumor suppressor, Singleminded 2s (SIM2s), expressed in breast epithelial cells that inhibits EMT and metastasis, and is downregulated in the progression of breast disease. Our previous studies found that loss of SIM2s expression results in downregulation of ESR1 and increased basal markers in the MCF7 (ER+BC) cell line. Likewise, Semaphorin 7a (SEMA7A) expression is associated with decreased overall survival in ER+BC patients. Patients with SEMA7Ahi tumors treated with Tamoxifen exhibit shortened relapse free survival. SEMA7A is a downstream target of both NFKB and PTGS2/COX-2; and our previously published results establish a cross-talk between NFKB and SIM2s to regulate PTGS2 via interactions with the AKT pathway. Moreover, COX-2 inhibition is associated with better prognosis for breast cancer patients. Therefore, we are investigating how SIM2s may repress AKT signaling in SEMA7A driven ER resistance and metastasis. Methods: We performed in silico analysis of the SEMA7A promoter using the ConTra web server. For in vitro assays, we examined estrogen dependent differences in SIM2s, ER, and SP1 binding utilizing luciferase reporter assays using intact promoter and SIM2s site deletion mutants (Invitrogen) in MCF7 cells. Using immunoblotting and qPCR analysis we also assessed expression of SEMA7A, ESR1, PI3K/AKT signaling genes and EMT associated genes in Crispr/Cas9 SIM2s knockout (SIM2KO) MCF7 cells. Treatment of MCF7 cells with exogenous SEMA7A was used to observe possible changes in SIM2s expression and SIM2s promoter activity. For in vivo assays, we used immunohistochemistry (IHC) to assess SIM2s in SEMA7A low and high expressing MCF7 xenografts. To examine the relationship between SIM2s and ER, SEMA7A-/- (KO) mice were crossed with C57/BL6 MMTV-PYMT mice. Results: In silico analysis of the SEMA7A promoter via ConTra revealed four potential SIM2 response elements containing the SIM2s central midline elements (CME) binding motif and highly conserved estrogen response element half-sites adjacent to two specificity protein 1 consensus bindings sites. Luciferase reporter assays in MCF7 cells confirmed SIM2s represses basal and estrogen induced-SEMA7A promoter activity. Moreover, SIM2s was unable to repress the SEMA7A promoter activity with CME mutation. MCF7 SIM2KO cells exhibit increased SEMA7A promoter activity, SEMA7A, AKT signaling, EMT signatures and decreased ESR1, CDH1, and PTEN protein and mRNA expression. Both SIM2s expression and SIM2s promoter activity are significantly decreased with the addition of exogenous SEMA7A. Moreover, MCF7 xenografts reveal reciprocal expression of SIM2s and SEMA7A. Additionally in MMTV-PyMT mice, SIM2s expression is lost as tumors transition from pre-malignant to invasive phenotypes, yet we observe maintenance of SIM2s and ER in SEMA7A-/-;PyMT mice. Conclusion: These findings establish a regulatory relationship between SIM2s and SEMA7A in ER+BC. SIM2s functions to downregulate SEMA7A mediated pro-tumor signaling and maintain ER expression, but can be turned off to allow for resistance in ER+BC. Given the observed reciprocal expression, our study suggests a SIM2s/SEMA7A switch which may confer resistance in ER+BC via AKT signaling through downregulation of PTEN. Thus, a SIM2s/SEMA7A switch may act as a prognostic indicator to provide therapeutic advantages in resistant ER+BC metastasis. Citation Format: Garhett L Wyatt, Lyndsey S Crump, Traci R Lyons, Weston W Porter. A SIM2s/SEMA7A switch drives therapeutic resistance in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-11.

Currently, immunotherapy with Bacillus Calmette-Guerin (BCG) is the most effective treatment for superficial bladder carcinoma, but treatment-related toxicity may limit its use in some patients. Alternative treatments are needed for patients who fail to respond to BCG immunotherapy. Allium sativum (AS), or garlic, is known to have a broad range of biologic activities, including immune stimulation and reported antitumor activity. For these reasons, the authors conducted a series of experiments designed to explore the possible therapeutic effects of AS in the MBT2 murine bladder carcinoma model. C3H/HeN mice were randomized prior to initiation of each experimental protocol. Mice received 1 x 10(3) MBT2 cells in 0.1 mL RPMI-1640, administered subcutaneously into the right thigh, on Day 0 of the experiment. AS was injected at the site of tumor transplantation on Day 1 and at 2- to 7-day intervals up to Day 28. To evaluate the effects of oral AS in this model, treatment was initiated 30 days prior to tumor inoculation and continued for 30 days after tumor inoculation. Animals in all experiments were followed for tumor incidence, tumor growth, and survival. In the initial experiments, subcutaneous AS significantly reduced tumor volume compared with the saline control (P < 0.05). Unfortunately, treatment-related death was also observed, requiring reduction in the total dose of AS. Animals that received 5 weekly immunizations of AS (5 mg, 5 mg, 1 mg, 1 mg, and 1 mg; cumulative dose = 13 mg) had significantly reduced tumor incidence, tumor growth, and increased survival when compared with animals that received the saline control. No treatment-related deaths were observed with this treatment schedule. To determine whether systemic AS administration might be effective, orally administered AS was tested at doses of 5 mg, 50 mg, and 500 mg per 100 mL of drinking water. Mice that received 50 mg oral AS had significant reductions in tumor volume (P < 0.05) when compared with animals that received the saline control, and mice that received 500 mg oral AS had significant reductions in both tumor volume and mortality (P < 0.05). The significant antitumor efficacy of subcutaneous and oral AS warrants further investigation and suggests that AS may provide a new and effective form of therapy for transitional cell carcinoma of the bladder.

&lt;p&gt;Materials and Methods Fig. S1. Time course of AUY922 treatment in BT-474 and MCF-7 cells. Fig. S2. Effects of AUY922 and PPP treatment in low and high HER2-expressing breast cancer cell lines. Fig. S3. Effect of HRG stimulation in AUY922 and PPP treated MCF-7 cells. Fig. S4. AUY922 and PPP treatment effect on the proliferative and invasive phenotype of BT-474 and MCF-7 breast cancer cells. Fig. S5. HPLC chromatograms of purified DFO-affibody conjugates. Fig. S6. MALDI-MS spectra of purified DFO-ZHER3:8698 (A) and DFO-ZTAQ (B). Fig. S7. Representative radio-ITLC of the crude radiolabelling mixture of 89Zr-DFO-ZHER3:8698. Fig. S8. Representative radio-ITLC of the 89Zr-DFO-ZHER3:8698 serum stability at 0, 3 and 24 h. Fig. S9. In vivo 89Zr-DFO-ZHER3:8698 tumour uptake quantification by image analysis. Fig. S10. Ex vivo biodistribution of 89Zr-DFO-ZHER3:8698 following AUY922 treatment in MCF-7 xenografts. Fig. S11. Microvessel density analysis in response to AUY922 treatment in MCF-7 xenografts. Fig. S12. Tumour volume estimation in response to AUY922 treatment in MCF-7 xenografts. Fig. S13. AUY922 treatment response monitoring in BT-474 xenografts by 89Zr-DFO-ZHER3:8698 PET/CT imaging. Fig. S14. Texture analysis of 89Zr-DFO-ZHER3:8698 PET imaging data following AUY922 treatment in MCF-7 xenografts. Fig. S15. Correlation between %ID/g ratios obtained from 89Zr-DFO-ZHER3:8698 PET images and HER3 protein expression per control and AUY922-treated MCF-7 xenografts. Fig. S16. HER3 intra-tumoral heterogeneity highlighted by histopathological analysis of control and AUY922-treated MCF-7 xenografts. Fig. S17. HER2-HER3 interaction in BT-474 and MCF-7 cells following AUY922 treatment. Fig. S18. Scheme illustrating the proposed effect of AUY922-induced Hsp90 inhibition in MCF-7 breast cancer xenografts. Table S1. Densitometric analysis of Western blots related to the treatment of BT-474, MCF-7, MCF-7 HER2+++ (-dox), and MCF-7 HER2+ (+dox) following 48 h treatment with AUY922 and PPP alone, or in combination. Table S2. Biodistribution results for 89Zr-DFO-ZHER3:8698 and 89Zr-DFO-ZTAQ (3 µg, 7.2-8.1 MBq/mouse) at 3 h after injection. Table S3. Biodistribution results for 89Zr-DFO-ZHER3:8698 at 3 h and 24 h after injection in MCF-7 and MDA-MB-468 tumour-bearing mice. Table S4. Tumour/organ ratios for 89Zr-DFO-ZHER3:8698 at 3 h and 24 h after injection in MCF-7 and MDA-MB-468 tumour-bearing mice. Table S5. HER3 and IGF1R co-occurrence in breast invasive carcinoma samples. Table S6. HER3 and IGF1R alterations lead to a shorter overall survival in breast invasive carcinoma samples. Table S7. HER3 and IGF1R alterations lead to a shorter disease free survival in breast invasive carcinoma samples. Table S8. HER3 and IGF1R co-occurrence in HER2 up-regulated breast invasive carcinoma samples.&lt;/p&gt;

&lt;p&gt;Materials and Methods Fig. S1. Time course of AUY922 treatment in BT-474 and MCF-7 cells. Fig. S2. Effects of AUY922 and PPP treatment in low and high HER2-expressing breast cancer cell lines. Fig. S3. Effect of HRG stimulation in AUY922 and PPP treated MCF-7 cells. Fig. S4. AUY922 and PPP treatment effect on the proliferative and invasive phenotype of BT-474 and MCF-7 breast cancer cells. Fig. S5. HPLC chromatograms of purified DFO-affibody conjugates. Fig. S6. MALDI-MS spectra of purified DFO-ZHER3:8698 (A) and DFO-ZTAQ (B). Fig. S7. Representative radio-ITLC of the crude radiolabelling mixture of 89Zr-DFO-ZHER3:8698. Fig. S8. Representative radio-ITLC of the 89Zr-DFO-ZHER3:8698 serum stability at 0, 3 and 24 h. Fig. S9. In vivo 89Zr-DFO-ZHER3:8698 tumour uptake quantification by image analysis. Fig. S10. Ex vivo biodistribution of 89Zr-DFO-ZHER3:8698 following AUY922 treatment in MCF-7 xenografts. Fig. S11. Microvessel density analysis in response to AUY922 treatment in MCF-7 xenografts. Fig. S12. Tumour volume estimation in response to AUY922 treatment in MCF-7 xenografts. Fig. S13. AUY922 treatment response monitoring in BT-474 xenografts by 89Zr-DFO-ZHER3:8698 PET/CT imaging. Fig. S14. Texture analysis of 89Zr-DFO-ZHER3:8698 PET imaging data following AUY922 treatment in MCF-7 xenografts. Fig. S15. Correlation between %ID/g ratios obtained from 89Zr-DFO-ZHER3:8698 PET images and HER3 protein expression per control and AUY922-treated MCF-7 xenografts. Fig. S16. HER3 intra-tumoral heterogeneity highlighted by histopathological analysis of control and AUY922-treated MCF-7 xenografts. Fig. S17. HER2-HER3 interaction in BT-474 and MCF-7 cells following AUY922 treatment. Fig. S18. Scheme illustrating the proposed effect of AUY922-induced Hsp90 inhibition in MCF-7 breast cancer xenografts. Table S1. Densitometric analysis of Western blots related to the treatment of BT-474, MCF-7, MCF-7 HER2+++ (-dox), and MCF-7 HER2+ (+dox) following 48 h treatment with AUY922 and PPP alone, or in combination. Table S2. Biodistribution results for 89Zr-DFO-ZHER3:8698 and 89Zr-DFO-ZTAQ (3 µg, 7.2-8.1 MBq/mouse) at 3 h after injection. Table S3. Biodistribution results for 89Zr-DFO-ZHER3:8698 at 3 h and 24 h after injection in MCF-7 and MDA-MB-468 tumour-bearing mice. Table S4. Tumour/organ ratios for 89Zr-DFO-ZHER3:8698 at 3 h and 24 h after injection in MCF-7 and MDA-MB-468 tumour-bearing mice. Table S5. HER3 and IGF1R co-occurrence in breast invasive carcinoma samples. Table S6. HER3 and IGF1R alterations lead to a shorter overall survival in breast invasive carcinoma samples. Table S7. HER3 and IGF1R alterations lead to a shorter disease free survival in breast invasive carcinoma samples. Table S8. HER3 and IGF1R co-occurrence in HER2 up-regulated breast invasive carcinoma samples.&lt;/p&gt;

Aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway supports growth of many tumors including those of breast, lung, and prostate. Resistance of breast cancer cells to targeted chemotherapies including tyrosine kinase inhibitors (TKI) has been linked to persistent PI3K activity, which may in part be due to increased membrane expression of epidermal growth factor (EGF) receptors (HER2 and HER3). Recently we found that proteins of the RGS (regulator of G protein signaling) family suppress PI3K activity downstream of the receptor by sequestering its p85alpha subunit from signaling complexes. Because a substantial percentage of breast tumors have RGS16 mutations and reduced RGS16 protein expression, we investigated the link between regulation of PI3K activity by RGS16 and breast cancer cell growth. RGS16 overexpression in MCF7 breast cancer cells inhibited EGF-induced proliferation and Akt phosphorylation, whereas shRNA-mediated extinction of RGS16 augmented cell growth and resistance to TKI treatment. Exposure to TKI also reduced RGS16 expression in MCF7 and BT474 cell lines. RGS16 bound the amino-terminal SH2 and inter-SH2 domains of p85alpha and inhibited its interaction with the EGF receptor-associated adapter protein Gab1. These results suggest that the loss of RGS16 in some breast tumors enhances PI3K signaling elicited by growth factors and thereby promotes proliferation and TKI evasion downstream of HER activation.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and its synthetic analog EB1089 induce characteristic morphological features of apoptosis in MCF-7 cells in vitro that coincide with up-regulation of clusterin and cathepsin B, proteins associated with apoptosis in the mammary gland, and with down-regulation of Bcl-2, an antiapoptotic protein. To determine whether vitamin D3 compounds could mediate apoptosis of breast tumors in vivo, we treated nude mice carrying established MCF-7 xenografts with the low calcemic vitamin D3 analog EB1089 via daily injection or sustained release pellets for up to 5 weeks. The volume of tumors from mice treated with 45 pmol/day EB1089 was 4-fold lower than that of tumors from vehicle-treated control mice after 5 weeks. The reduced growth of tumors from EB1089-treated mice was associated with characteristic apoptotic morphology and a marked reduction in the proportion of epithelial cells to stroma. After 5 weeks of treatment with EB1089, MCF-7 tumors exhibited a 6-fold increase in DNA fragmentation (as measured by in situ end labeling) relative to that in control tumors. The enhanced rate of apoptosis in tumors from EB1089-treated mice was coupled to a 2-fold reduction in proliferation (as measured by expression of proliferating cell nuclear antigen) compared with that in tumors from control mice. The antitumor effects of EB1089 were evident at doses that had minimal effects on serum calcium and body weight. EB1089 treatment did not alter the growth of xenografts derived from a vitamin D3-resistant variant of MCF-7 cells (MCF-7(D3Res) cells), which display resistance to EB1089 in vitro, indicating that resistance to EB1089 is maintained in vivo. Tumors derived from both MCF-7 and MCF-7(D3Res) cells underwent apoptotic regression upon estradiol withdrawal, indicating comparable estrogen dependence of tumors with differential sensitivity to vitamin D3 compounds. These are the first studies to demonstrate apoptotic morphology and regression of human breast tumors in response to treatment with a vitamin D3 analog in vivo and support the concept that vitamin D3 compounds can effectively target human breast cancer.

Platinum complexes inhibit HER-2 enriched and triple-negative breast cancer cells metabolism to suppress growth, stemness and migration by targeting PKM/LDHA and CCND1/BCL2/ATG3 signaling pathways.