Biophysical Phenotyping and Modulation of ALDH+ Inflammatory Breast Cancer Stem-Like Cells.

Cancer stem-like cells (CSCs) have been shown to initiate tumorigenesis and cancer metastasis in many cancer types. Although identification of CSCs through specific marker expression helps define the CSC compartment, it does not directly provide information on how or why this cancer cell subpopulation is more metastatic or tumorigenic. In this study, the functional and biophysical characteristics of aggressive and lethal inflammatory breast cancer (IBC) CSCs at the single-cell level are comprehensively profiled using multiple microengineered tools. Distinct functional (cell migration, growth, adhesion, invasion and self-renewal) and biophysical (cell deformability, adhesion strength and contractility) properties of ALDH+ SUM149 IBC CSCs are found as compared to their ALDH- non-CSC counterpart, providing biophysical insights into why CSCs has an enhanced propensity to metastasize. It is further shown that the cellular biophysical phenotype can predict and determine IBC cells' tumorigenic ability. SUM149 and SUM159 IBC cells selected and modulated through biophysical attributes-adhesion and stiffness-show characteristics of CSCs in vitro and enhance tumorigenicity in in vivo murine models of primary tumor growth. Overall, the multiparametric cellular biophysical phenotyping and modulation of IBC CSCs yields a new understanding of IBC's metastatic properties and how they might develop and be targeted for therapeutic interventions.

MMP2 and MMP9 serum levels are associated with favorable outcome in patients with inflammatory breast cancer treated with bevacizumab-based neoadjuvant chemotherapy in the BEVERLY-2 study.

PURPOSE: Addition of bevacizumab to trastuzumab-based neoadjuvant chemotherapy in HER2-positive inflammatory breast cancer (IBC) was associated with favorable outcome in the BEVERLY-2 phase II trial. Circulating levels of matrix metalloproteinases (MMP) 2 and 9 were correlated to high response rate and prolonged survival in high-grade glioma treated with bevacizumab. We examined the prognostic impact of MMP2 and MMP9 serum levels in BEVERLY-2 patients. EXPERIMENTAL DESIGN: MMP2 and MMP9 serum levels were assessed using ELISA at baseline and before surgery in 45/52 available samples. Correlations were tested with pathological complete response (pCR), disease-free survival (DFS) and overall survival (OS). RESULTS: Baseline (b) MMP2 and MMP9 serum levels were independent from patient characteristics and circulating tumor or endothelial cells, and were not correlated to pCR. High bMMP2 was correlated to better DFS (p=0.001) and OS (p=0.032), while low bMMP9 was correlated to better OS (p=0.022) and tended to be associated with longer DFS (p=0.071). In multivariate analyses, bMMP2 (p=0.003, Hazard Ratio [HR]: 0.115) and bMMP9 (p=0.041, HR: 3.511) remained correlated to DFS. As continuous variables, bMMP2 was associated with relapse (p=0.002) and death (p=0.049), while bMMP9 was associated with death (p=0.035). During treatment, significant increase in MMP2 and decrease in MMP9 levels (p<0.001 for both) were observed in 100% and 87% of patients respectively. CONCLUSIONS: High bMMP2 and low bMMP9 serum levels were associated with better survival in HER2-positive IBC patients treated with bevacizumab- and trastuzumab-based neoadjuvant chemotherapy. Their predictive value of bevacizumab benefit should be evaluated in a randomized trial.

HDAC6 activity is a non-oncogene addiction hub for inflammatory breast cancers.

INTRODUCTION: Inflammatory breast cancer (IBC) is the most lethal form of breast cancers with a 5-year survival rate of only 40 %. Despite its lethality, IBC remains poorly understood which has greatly limited its therapeutic management. We thus decided to utilize an integrative functional genomic strategy to identify the Achilles' heel of IBC cells. METHODS: We have pioneered the development of genetic tools as well as experimental and analytical strategies to perform RNAi-based loss-of-function studies at a genome-wide level. Importantly, we and others have demonstrated that these functional screens are able to identify essential functions linked to certain cancer phenotypes. Thus, we decided to use this approach to identify IBC specific sensitivities. RESULTS: We identified and validated HDAC6 as a functionally necessary gene to maintain IBC cell viability, while being non-essential for other breast cancer subtypes. Importantly, small molecule inhibitors for HDAC6 already exist and are in clinical trials for other tumor types. We thus demonstrated that Ricolinostat (ACY1215), a leading HDAC6 inhibitor, efficiently controls IBC cell proliferation both in vitro and in vivo. Critically, functional HDAC6 dependency is not associated with genomic alterations at its locus and thus represents a non-oncogene addiction. Despite HDAC6 not being overexpressed, we found that its activity is significantly higher in IBC compared to non-IBC cells, suggesting a possible rationale supporting the observed dependency. CONCLUSION: Our finding that IBC cells are sensitive to HDAC6 inhibition provides a foundation to rapidly develop novel, efficient, and well-tolerated targeted therapy strategies for IBC patients.

HER2 as a predictive factor for successful neoadjuvant anthracycline chemotherapy of locally advanced and early breast cancer.

OBJECTIVE: The aim of this study was to look for predictive and prognostic factors in anthracycline-based neoadjuvant chemotherapy. METHODS: Three hundred and nine patients with early-stage or locally advanced breast cancer were enrolled in this study and preoperatively treated with neoadjuvant chemotherapies in intense dose-dense (cyclophosphamide + epirubicin) or conventional (paclitaxel + epirubicin) regimens. Outcome parameters included overall objective response rate, as well as factors for determining pathological features influencing the efficacy of chemotherapy, such as estrogen receptor, progesterone receptor, and HER2 status, Eastern Cooperative Oncology Group (ECOG) score, tumor size, tumor inflammation, and lymph node metastasis. RESULTS: The overall pathological complete response (pCR) rate was 14.3%, and the main factor affecting the efficacy of chemotherapy was HER2 expression. The pCR rate was significantly higher for patients with HER2 overexpression, compared with patients with low HER2 expression (27.5% vs. 9.6%, p<0.001). The recurrence risk for patients with pCR decreased 1.12-fold compared with patients without pCR (5-year disease-free survival [DFS]: 81.8% vs. 65.7%, p=0.038). Patients in the HER2 overexpression subgroup benefited more from pCR than those in the HER2 low expression group (5-year DFS: 86.4% vs. 62.1%, p=0.049). CONCLUSION: HER2 overexpression in primary tumors might be a predictive marker for good efficacy of anthracycline-based neoadjuvant chemotherapy. The pCR is a suitable prognostic factor, even for patients with HER2 overexpression.

Aldehyde dehydrogenase 1 expression in inflammatory breast cancer as measured by immunohistochemical staining.

BACKGROUND: Inflammatory breast cancer (IBC) is a rare but aggressive type of breast carcinoma. Despite multimodality approaches, the clinical outcome of patients with IBC remains poor. Tumors arising from cancer stem cells (CSCs) are associated with drug resistance, tumor recurrence, and poor prognosis. This study aimed to evaluate expression of aldehyde dehydrogenase 1 (ALDH1), a putative stem cell marker, in IBC tumors. MATERIALS AND METHODS: Tissue microarrays of 74 surgically resected IBC tumors were immunohistochemically stained for ALDH1. The results were correlated with clinicopathologic parameters and survival data and were compared with findings published in the literature. RESULTS: The median follow-up time of the cohort was 42.1 months, and the 5-year overall survival (OS) rate was 46%. Twenty-four tumors (32%) were positive for ALDH1 staining. However, ALDH1 expression was not significantly associated with clinicopathologic variables, including lymph node status, tumor grade, and the status of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Log-rank testing found that ALDH1 expression was not significantly associated with the OS rate, although there was a trend toward an association with lower OS rate (P = .07). The findings were consistent with some of the published data, but substantial inconsistency among reports was noted. CONCLUSION: In this IBC cohort, no significant correlation between ALDH1 expression and prognosis or other clinicopathologic variables was found. Although sample size and selection criteria may be contributory factors, inconsistent results reported in the literature raise concern regarding the reliability of immunohistochemically identified ALDH1 as a sole marker of breast CSCs. Further study is required to elucidate the significance of CSCs in IBC biology.

Regulation of inflammatory breast cancer cell invasion through Akt1/PKBα phosphorylation of RhoC GTPase.

With a 42% and 18% 5- and 10-year respective disease-free survival rate, inflammatory breast cancer (IBC) is arguably the deadliest form of breast cancer. IBC invades the dermal lymphatic vessels of the skin overlying the breast and as a consequence nearly all women have lymph node involvement and ~1/3 have gross distant metastases at the time of diagnosis. One year after diagnosis ~90% of patients have detectable metastases, making IBC a paradigm for lymphovascular invasion. Understanding the underlying mechanisms of the IBC metastatic phenotype is essential for new therapies. Work from our laboratory and others show distinct molecular differences between IBC and non-IBCs (nIBCs). Previously we showed that RhoC GTPase is a metastatic switch responsible for the invasive phenotype of IBC. In this study we integrate observations made in IBC patients with in vitro analysis. We show that the PI3K/Akt signaling pathway is crucial in IBC invasion. Key molecules involved in cytoskeletal control and cell motility are specifically upregulated in IBC patients compared with stage and cell-type-of-origin matched nIBCs patients. Distinctively, RhoC GTPase is a substrate for Akt1 and its phosphorylation is absolutely essential for IBC cell invasion. Further our data show that Akt3, not Akt1 has a role in IBC cell survival. Together our data show a unique and targetable pathway for IBC invasion and survival.

Farnesyl transferase inhibitor treatment of breast cancer cells leads to altered RhoA and RhoC GTPase activity and induces a dormant phenotype.

Farnesyl transferase inhibitors (FTIs) were shown to be effective in modulating tumor growth in Ras-transformed tumor cells. Recent studies have focused on Rho GTPases as putative targets of FTI action. Previously, we demonstrated that FTIs were effective in inhibiting the growth and invasiveness of RhoC GTPase-overexpressing inflammatory breast cancer (IBC) cells however, RhoC activity was increased. In this study, we examine the mechanisms of FTI action on breast cancer cells in culture through modulation of RhoC and RhoA GTPases. We found that FTI inhibition of breast cancer cell growth was reversible and resembled what has been described for an in vitro model of tumor cell dormancy. On FTI treatment, levels of active RhoA decreased significantly, whereas levels of active RhoC increased 3.8-fold. We studied the role of these two GTPases in a fibronectin and basic FGF-induced model of breast cancer cell dormancy. Hypoactivation of RhoA and hyperactivation of RhoC were seen to induce morphology and growth changes consistent with tumor cell dormancy in culture. In addition, the JNK/SAPK pathway was induced on FTI treatment. A pharmacologic inhibitor of the JNK/SAPK pathway significantly reduced the number of dormant cells. This study has implications for the use of FTIs as therapeutic agents as well as potential mechanisms for breast cancer cell dormancy.

CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy.

Malignant transformation and maintenance of the malignant phenotype depends on oncogenic and non-oncogenic proteins that are essential to mediate oncogene signaling and to support the altered physiologic demands induced by transformation. Protein kinase CK2 supports key prosurvival signaling pathways and represents a prototypical non-oncogene. In this study, we describe CX-4945, a potent and selective orally bioavailable small molecule inhibitor of CK2. The antiproliferative activity of CX-4945 against cancer cells correlated with expression levels of the CK2α catalytic subunit. Attenuation of PI3K/Akt signaling by CX-4945 was evidenced by dephosphorylation of Akt on the CK2-specific S129 site and the canonical S473 and T308 regulatory sites. CX-4945 caused cell-cycle arrest and selectively induced apoptosis in cancer cells relative to normal cells. In models of angiogenesis, CX-4945 inhibited human umbilical vein endothelial cell migration, tube formation, and blocked CK2-dependent hypoxia-induced factor 1 alpha (HIF-1α) transcription in cancer cells. When administered orally in murine xenograft models, CX-4945 was well tolerated and demonstrated robust antitumor activity with concomitant reductions of the mechanism-based biomarker phospho-p21 (T145). The observed antiproliferative and anti-angiogenic responses to CX-4945 in tumor cells and endothelial cells collectively illustrate that this compound exerts its antitumor effects through inhibition of CK2-dependent signaling in multiple pathways. Finally, CX-4945 is the first orally bioavailable small molecule inhibitor of CK2 to advance into human clinical trials, thereby paving the way for an entirely new class of targeted treatment for cancer.