Presence of anaplastic lymphoma kinase in inflammatory breast cancer

Although Inflammatory Breast Cancer (IBC) is recognized as the most metastatic variant of locally advanced breast cancer, the molecular basis for the distinct clinical presentation and accelerated program of metastasis of IBC is unknown. Reverse phase protein arrays revealed activation of the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK) and biochemically-linked downstream signaling molecules including JAK1/STAT3, AKT, mTor, PDK1, and AMPK\uce\ub2 in pre-clinical models of IBC. To evaluate the clinical relevance of ALK in IBC, analysis of 25 IBC patient tumors using the FDA approved diagnostic test for ALK genetic abnormalities was performed. These studies revealed that 20/25 (80%) had either increased ALK copy number, low level ALK gene amplification, or ALK gene expression, with a prevalence of ALK alterations in basal-like IBC. One of 25 patients was identified as having an EML4-ALK translocation. The generality of gains in ALK copy number in basal-like breast tumors with IBC characteristics was demonstrated by analysis of 479 breast tumors using the TGCA data-base and our newly developed 79 IBC-like gene signature. The small molecule dual tyrosine kinase cMET/ALK inhibitor, Crizotinib (PF- 02341066/Xalkori\uc2\uae, Pfizer Inc), induced both cytotoxicity (IC50= 0.89 \uce\ubcM) and apoptosis, with abrogation of pALK signaling in IBC tumor cells and in FC-IBC01 tumor xenograft model, a new IBC model derived from pleural effusion cells isolated from an ALK+IBC patient. Based on these studies, IBC patients are currently being evaluated for the presence of ALK genetic abnormalities and when eligible, are being enrolled into clinical trials evaluating ALK targeted therapeutics. \uc2\ua9 2013 Robertson et al

Abstract 4988: Clues for targeted therapies in inflammatory breast cancer (IBC)

Abstract Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer characterized by rapid proliferation, early metastatic development, and poor prognosis. The peculiar clinical presentation of IBC characterized by breast erythema and swelling is caused by the invasion of aggregates of tumor cells (tumor emboli) into the dermal lymphatics, causing an obstruction of the lymph channels. Although, tumor emboli are also occasionally demonstrated in non-IBC tumors, they are more frequently detected and more numerous in IBC. Tumor emboli expressed cell-cell adhesion molecules that maintain the tumor cells together. The high risk of disease recurrence in soft tissue and lymph nodes shortly after completion of multidisciplinary treatment particularly in patients with residual disease supports the hypothesis that these tumor emboli are critically related to the development of micro metastatic disease. We developed a new model of IBC derived from the pleural effusion of a woman with metastatic secondary IBC. FC-IBC02 cells are triple negative and form clusters in suspension that were strongly positive for E-cadherin, β-catenin, and TSPAN24, all adhesion molecules that play an important role in cell migration and invasion. The maintenance of cell-cell adhesions allow the migration of tumor cells through the lymphatic and blood vessels as clusters. FC-IBC02 cells shown a partial or incomplete epithelial-mesenchymal transition (EMT); these cells express some epithelial markers (EpCAM, E-cadherin) and also mesenchymal markers (VIM, FN1, Snai2, Twist1). Furthermore, circulating tumor cells (CTC) from IBC patients are present in the blood as single cells and clusters supporting the collective migration of tumor cells in IBC. FC-IBC02 cells were highly metastatic when injected in the fat mammary pad of SCID mice and these cells were able to produce brain metastasis in mice by intracardiac or intra-peritoneal injections. Genomic studies of FC-IBC02 and other IBC cell lines showed that IBC cells had important amplification of 8q24 where MYC, ATAD2 and the focal adhesion kinase FAK1 are located. MYC and ATAD2 showed between 2.5-7 copies in IBC cells. FAK1, which plays important roles in anoikis resistance and tumor metastasis, showed 6-4 copies in IBC cells. FAK1 is amplified, upregulated and phosphorylated (active) in inflammatory breast cancer. Additionally, FC-IBC02 showed amplification of ALK and NOTCH3. Our results indicate that MYC, ATAD2, CD44, NOTCH3, ALK and/or FAK1 may be used as potential targeted therapies against IBC. We are currently evaluating an ALK/FAK inhibitor using the novel established IBC model. Citation Format: Sandra V. Fernandez, Fredika M. Robertson, Sankar Addya, Zhaomei Mu, Massimo Cristofanilli. Clues for targeted therapies in inflammatory breast cancer (IBC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4988. doi:10.1158/1538-7445.AM2014-4988</jats:p

MR-guided focused ultrasound: enhancement of intratumoral uptake of [³H]-docetaxel in vivo

The purpose of this study is to quantify the enhancement of [³H]-docetaxel in implanted prostate tumors treated with MR-guided pulsed focused ultrasound (MRgFUS). Human prostate cancer, LNCaP cells in 25 µl, were implanted into the prostates of male nude mice. The tumor growth was directly monitored on MRI. When the tumor reached a designated size, MRgFUS treatment was performed using a focused ultrasound treatment system (InSightec ExAblate 2000) with a 1.5 T GE MR scanner. The tumor-bearing animals were randomly divided into three groups: group 1, MRgFUS treatment + [³H]-docetaxel; group 2, [³H]-docetaxel only and group 3, as a control. Animals in group 1 were treated with MRgFUS non-invasively. Immediately after the treatment, the animals received a single dose of tail vein injection of docetaxel at 15 mg kg⁻¹ mixed with [³H]-docetaxel at 50 uCi kg⁻¹ in a total volume of 150 µl. Animals in group 2 were treated the same as in group one, however without MRgFUS treatment. Animals in group 3 were treated as a control. Animals were sacrificed 30 min after i.v. injections regardless of whether or not they received focused ultrasound. Tumors were removed and processed. The radioactivity of [³H]-docetaxel in the tumor tissue was quantitatively measured by a liquid scintillation counter. Our study showed that all animals tolerated the MRgFUS treatment well. Our data showed increased (³H-docetaxel concentration in the tumor in the MRgFUS-treated group (1079 ± 132 cmp/75 mg) versus those without MRgFUS treatment (524 ± 201 cmp/75 mg) with P = 0.037

Antisense-MDM2 Sensitizes LNCaP Prostate Cancer Cells to Androgen Deprivation, Radiation, and the Combination In Vivo

Purpose: To test the effects of antisense (AS)-MDM2 alone and with androgen deprivation (AD), radiotherapy (RT), and AD + RT on wild-type LNCaP cells in an orthotopic in vivo model. Methods: Androgen-sensitive LNCaP cells were grown in the prostates of nude mice. Magnetic resonance imaging–based tumor volume and serum prostate-specific antigen (PSA) measurements were used to assess effects on tumor response. Tumor response was measured by biochemical and tumor volume failure definitions and doubling time estimates from fitted PSA and tumor volume growth curves. Expression of MDM2, p53, p21, and Ki-67 was quantified using immunohistochemical staining and image analysis of formalin-fixed tissue, analogous to methods used clinically. Results: Antisense-MDM2 significantly inhibited the growth of LNCaP tumors over the mismatch controls. The most significant increase in tumor growth delay and tumor doubling time was from AS-MDM2 + AD + RT, although the effect of AS-MDM2 + AD was substantial. Expression of MDM2 was significantly reduced by AS-MDM2 in the setting of RT. Conclusions: This is the first in vivo investigation of the effects of AS-MDM2 in an orthotopic model and the first to demonstrate incremental sensitization when added to AD and AD + RT. The results with AD underscore the potential to affect micrometastatic disease, which is probably responsible for treatment failure in 30–40% of men with high-risk disease

Edelfosine Promotes Apoptosis in Androgen-Deprived Prostate Tumors by Increasing ATF3 and Inhibiting Androgen Receptor Activity

Edelfosine is a synthetic alkyl-lysophospholipid (ALP) that possesses significant antitumor activity in several human tumor models. Here, we investigated the effects of edelfosine combined with androgen deprivation (AD) in LNCaP and VCaP human prostate cancer cells. This treatment regimen greatly decreased cell proliferation compared to single agent or AD alone resulting in higher levels of apoptosis in LNCaP compared to VCaP cells. Edelfosine caused a dose-dependent decrease in AKT activity, but did not affect the expression of total AKT in either cell line. Furthermore, edelfosine treatment inhibited the expression of androgen receptor (AR) and was associated with an increase in activating transcription factor 3 (ATF3) expression levels, a stress response gene and a negative regulator of AR transactivation. ATF3 binds to AR after edelfosine + AD and represses the transcriptional activation of AR as demonstrated by prostate specific antigen (PSA) promoter studies. Knockdown of ATF3 using siRNA-ATF3 reversed the inhibition of PSA promoter activity, suggesting that the growth inhibition effect of edelfosine was ATF3 dependent. Moreover, expression of AR variant 7 (ARv7) and TMPRSS2-ERG fusion gene were greatly inhibited after combined treatment with AD and edelfosine in VCaP cells. In vivo experiments using an orthotopic LNCaP model confirmed the anti-tumor effects of edelfosine + AD over the individual treatments. A significant decrease in tumor volume and PSA levels were observed when edelfosine and AD were combined, compared to edelfosine alone. Edelfosine shows promise in combination with AD for the treatment of prostate cancer patients

Fabrication of a Near-Infrared Upconversion Nanosensor for the Ultrasensitive Detection of eARGs Using a Dual-Amplification Strategy

Extracellular antibiotic resistance genes (eARGs) have emerged as significant environmental contaminants due to their role in the transmission and proliferation of antibiotic-resistant bacteria, posing a major threat to global health. Traditional detection methods for eARGs require complicated equipment, lengthy analysis times, and relatively low selectivity. Furthermore, eARGs are present in low concentrations in surface water samples, which presents considerable challenges to the sensitivity of detection assays. Therefore, there is an urgent need to develop more accessible, stable, and sensitive detection methods. In this work, we developed an ultrasensitive upconversion nanosensor utilizing a dual-amplification strategy for the detection of trace eARGs (bla-TEM). The upconversion nanosensor was activated upon the capture of bla-TEM and subsequently enriched through magnetic separation. Following this, a cascade nicking-polymerization amplification process occurred in a single reaction facilitated by a magnetic capture probe, an upconversion recognition probe, and the relevant enzymes. The upconversion nanosensor functions as both the direct target-recognizing moieties and signal reporters, replacing the energy donor in conventional luminescence resonance energy transfer-based upconversion nanosensors. Ultimately, the strategy demonstrated excellent sensitivity with a limit of detection (LOD) of 0.093 aM, rapid detection in less than one hour, good selectivity, and high accuracy compared to conventional polymerase chain reaction (PCR) assays. These findings provide valuable insights for the development of ultrasensitive detection assays for emerging environmental pollutants