Microarray Analysis of Induced Inflammatory Gene Changes with Doxorubicin and a Second-generation Proteasome Inhibitor in Human Triple Negative MDA_MB-231 Breast Cancer Cell Line

Second-generation Proteasome Inhibitor Human Triple Negative MDA-MB-231 Abstract Purpose: Inflammation is well known to be a key culprit behind tumor promotion and metastasis in Triple Negative Breast Cancer. In hopes of further understanding TNBC pro-inflammatory drug effects, we have studied the current first-line therapy drug doxorubicin along with a second-generation proteasome inhibitor that demonstrate a reduction in inflammation. In this study, we focused on conducting a gene array to elucidate the effects of carfilzomib and doxorubicin on

. The resultant secretion of chemokines leads to increased cell motility, invasiveness, and survival in response to inflammation Mantovani et al. [2]. This is especially prominent in TNBC.
Several first line chemotherapeutic drugs target TNBC, along with many other cancers, -Doxorubicin being one that is more commonly used. Studies have demonstrated that doxorubicin, although potent, further induces inflammatory processes Lopez-Hisijos N [3]. Doxorubicin also exhibits high short and long-term morbidity and mortality with severe heart failure occurring in more than 20% of patients Stagg and Allard [4]. In an effort to reduce these toxic effects, there is a lifetime dose limit; however, this commonly leads to under treatment of patients and eventual recurrence. This drug increases production of Transforming Growth Factor Beta (TGFB), leading to epithelial-mesenchymal transition and subsequent cell invasion in human MDA-MB-231 cell lines Bandyopadhyay et al. [5]; Vyas D [6]. Thus, a better understanding of the role of inflammation in TNBC treatment is imperative.
Carfilzomib (CARF) is a second-generation epoxiketone-based proteasome inhibitor that binds irreversibly to the 20S proteolytic core of the 26S proteasome Riz et al. [7]. Proteasome inhibitors are known to trigger anti-inflammatory effects within various cell lineages. The ability of CARF to not only kill tumor cells but also block the inflammatory pathways characteristic of TNBC suggests that CARF might have an effective therapeutic role in this disease.
By utilizing CARF alongside doxorubicin, we are able to understand on a molecular level their role in TNBC, and further explore their mode of action on gene regulation.
In this current study, we focused on identifying the effects

Quantitative Pcr Confirms Gene Array Expression Data of Select Genes
Prior to analysis all purified RNA was checked for quality on an Agilent BioAnalyzer. Samples meeting the quality criteria, an RNA Integrity Number (RIN) score of 8 or above were chosen for analysis. RNA samples were first tested for residual genomic DNA contamination by performing QPCR using the TWIST gene assay (which is able to detect genomic DNA) and 2ng of RNA as  Tables 1-2 display the selected genes for this study and also include control cDNAs of the housekeeping gene such as 18S for signal normalization and quantitation. Genes that were up-regulated by more than 1.5-fold or down-regulated by at least 1.5-fold were considered significant and a comparison of fold-change in expression of these controls, MCF-10A versus MDA-MB-231, was made (Tables 1A and 1B). In addition, a more comparative analysis and categorization of gene expression were made based on similarities and differences in individual expression profiles (Table 2).     (Table 2).

Discussion
In this experiment, we found many inflammatory genes to

Carfilzomib Induces Down-Regulation of Pro-Inflammatory Gene Klkb1
Amongst the genes studied and targeted in our experiment, we observed the unexpected change in regulation of two novel genes previously unmentioned in the literature. The enzyme encoded by KLKB1 is known to be involved in a pro-inflammatory cascade, ultimately resulting in the release of Bradykinin Matsumoto et al. [9]. Bradykinin promotes inflammation by increasing blood vessel permeability and thus serves as a potential trigger for enhanced metastasis. HPGD, on the other hand, encodes an anti-inflammatory enzyme that metabolizes prostaglandin Lee et al. [10]. In our study,

Carfilzomib and Doxorubicin Induce Down-Regulation of Pro-Inflammatory Gene Vcam1
There were two specific genes that consistently showed increased expression in MDA-MB-231 cells; TNFSF13B and VCAM1.
The former is a cytokine expressed in B cells and is also a potent activator Gene 2015b [11]. It has been found to be constantly expressed in adipocytes of normal breast cells and promotes the proliferation and development of normal as well as malignant breast tissue Pelekanou et al. [12]. The latter is an NF-kB related gene that is responsible for encoding a cell surface protein that mediates leukocyte-endothelial cell adhesion. Its up regulation is tumor-promoting, particularly in inflammatory breast cancer Lerebours et al. [13]. In addition, VCAM1 also plays a significant Banks et al. [17].

Carfilzomib and Doxorubicin Induce Il1rapl2 Gene Suppression
In the MCF-10A normal breast epithelial cell line, we found consistently increased gene expression with PTAFR and IL1RAPL2.
PTAFR serves as a binding site for platelet activating factor, which is a phospholipid that plays a significant role in inflammation, and its production in MDA-MB-231 cells has been correlated to angiogenesis, increased cell motility, and proliferation Bussolati et al. [18]; Gene 2015a [19]. IL1RAPL2 is a receptor for an interleukin 1 accessory protein that when activated, facilitates signal transduction pathways. IL-1 is a cytokine with various functions in both physiological and pathological states and is known to be up regulated in many tumor types including breast tumors Lewis et al. [14]. cells. This is supported and confirmed by multiple studies conducting gene profiles on inflammatory breast cancer subtypes Bertucci et al. [21]; Sorlie et al. [22]; Van Laere et al. [23]. Combined with our analysis of fold-changes after treatment with carfilzomib and doxorubicin, it is notable that inflammation levels can be decreased even further upon addition of the second-generation proteasome inhibitor. More anti-inflammatory results were seen with the down regulation of KLKB1, VCAM1, and IL1RAPL2, all of which are known to be up involved in TNBC (Vyas D) [24,25]. Similarly, targeting and minimizing the up-regulation of HPGD, TNFSF13B and PTAFR can potentially further reduce inflammation-induced toxicity and metastasis. The increased downregulation of genes seen with carfilzomib highlights its potential to be used either as a standalone or combinational drug alongside with doxorubicin. Further studies such as gene silencing or knockout are required to fully understand the role and mechanism of carfilzomib and doxorubicin on the TNBC inflammatory disease profile. However, it is evident that a supplementary or alternative strategy is necessary to ameliorate the negative effects of TNBC prognosis. Our microarray study serves as a preliminary insight towards an enhanced regimen for tumor-suppression in TNBC [26,27].