Precise targeting of the molecular factors associated with cancer pathogenesis is prerequisite for an effective cure. For this purpose, identification of novel therapeutic targets and development of corresponding molecules is a continuous process. ACGs are endogenous enemies of transformed cells and are being investigated for their prognostic and therapeutic relevance. Expressional profiling of various ACGs in cancers, their association with disease progression and involvement in treatment response is being studied by the scientific community. In parallel, novel delivery methods including antibody-based conjugates, immunotoxins development and genetic engineering strategies are being established to achieve ACGs mediated cancer cell-specific death. In addition to these synthetic strategies, naturally occurring compounds are also being considered for their potential to induce ACGs in cancer cells and to exploit resulting antitumor effects.
In this study, as a first step, we identified the protein expression levels of three ACGs (NOXA, PAR-4, TRAIL) in breast cancer tissues by immunohistochemistry techniques (Fig. 1). To the best of our knowledge, this is the first study to investigate the expression profile of multiple ACGs at a time in breast cancer patients. At this time, we focused on IDC samples only, which is the most prevalent type of breast cancer. As far as the patient cohort is concerned, early onset of the disease (average ± 44 years age) with a considerable proportion of young patients (n = 20/45: <40 years age) was noticed, which is mainly attributed to the lack of awareness programs and late diagnosis locally. Molecular subtyping revealed a maximum number of cases as triple negative (Basal like) followed by Luminal A type, which itself predicts poor prognosis of these patients. As far as immunohistochemistry results are concerned, distinct ACG expression profiles were identified in breast cancer patient samples. An almost even distribution of NOXA expression (Neg/Low 21/45 = 47%, Elevated 24/45 = 53%) was found with no correlation with the molecular subtypes of breast cancer. However, a clear trend was found between NOXA expression and tumor grade, where Neg/Low expression ratios increased with increasing grade of the disease (Table 2A). This, in turn, reflects continuous inhibition of NOXA expression overtime in patients with advanced stages of breast cancer. Similar findings have also been reported where low expression of NOXA was found associated with poor prognosis, reduced overall survival and chemo-resistance in breast cancer [6, 7]. In our selected cohort, low expression of the PAR-4 gene was a dominant fact where 76% (34/45) of the samples were found negative for this ACG. Furthermore, a continuous decrease in PAR-4 expression was witnessed with the increasing severity of the disease. Precisely, 4/8 (50%), 12/16 (75%) and 18/21 (86%) cases of grade 1, 2 and 3 were found negative for PAR-4 expression, respectively (Table 2A). The data is in line with other available reports where low expression of PAR-4 and its association with poor prognosis is reported in breast cancer [9, 8]. Lower expression of PAR-4 was also correlated significantly (p < 0.05) with the luminal A and basal type of breast carcinomas, thus depicting the poor response to treatment and an unfavourable outcome. Similar findings have been reported, where PAR-4 expression was found to be low in the highly aggressive, estrogen-receptor negative (ER-), basal-like, and high-grade (grade 3) breast cancers, which are all associated with poor clinical outcome [33]. Alvarez and colleagues found lower PAR-4 expression associated with the poor response towards neoadjuvant chemotherapy and an increased risk of relapse in patients with breast cancer. Concurrently, higher expression of PAR-4 is also reported to sensitize triple-negative breast cancer cell lines to DNA damage-induced cell death by making cells more prone to chemo-sensitivity [34]. All in all, our data and available scientific reports suggest overall low expression of PAR-4 in breast cancer patients with a substantial association with aggressive nature, chemo-resistance and relapse of the disease. TRAIL is one of the most studied ACG till today and is being targeted in the field of cancer therapeutics. A major hurdle is to overcome the development of TRAIL resistance achieved by tumour cells. Despite these existing challenges, the development of specific molecules (agonists) against the TRAIL receptors and recombinant TRAIL is an on-going process. As far as the expression profile is concerned, TRAIL was the only gene where we found positive expression (at least 10% of tumour cells positively stained) in most of the clinical samples (32/45 = 71%, Table 1B). This positivity of the expression was increased with the severity of the disease as shown by 3/8 (38%), 11/16 (69%) and 16/21 (76%) cases in grade 1, 2 and 3, respectively (Table 2A). Higher levels of TRAIL correlated significantly (p < 0.05) with the luminal A and basal type of breast carcinomas. Grade 3 cases in the present study demonstrated a significant association with higher TRAIL expression and negative PAR-4 expression thus depicting that these two markers relate significantly with tumour aggressiveness and poor prognosis in breast cancer patients (Table 2A). Over and above to these facts, astonishing findings are being reported where combination of TRAIL along with platinum drugs (cisplatin) and liposomal formulations have shown to be effective against cancer stem cells and circulating breast cancer cells, which in turn, gives us hope that exploiting ACGs can be effective in controlling secondary tumour development and recurrence of the disease [12, 35].
Several natural compounds have shown the potential to up-regulate ACGs in cancer cells followed by induction of multidirectional antineoplastic effects [36–40]. As we know, by enlarge naturally occurring agents bear a safe physiological profile, thus exploiting such compounds to up-regulate ACGs in cancer cells seems to be an attractive research domain. Among the plant-based anticancer agents, RIPs comprise an important class of toxins with the promising capacity to be developed as therapeutic entities [41]. The potential effects of RIPs’ exposure on endogenous enemies of cancer cells (ACGs) are a neglected field so far. In a previous study, we reported that riproximin can induce significantly (˃100fold) the expression of a well-known ACG (MDA-7/IL24) in breast cancer cells [26]. In this particular study, we were interested to figure out the potential impact of riproximin on expressional profile of three further ACGs (NOXA, PAR-4, TRAIL). For this purpose, two breast cancer cell lines with distinct molecular subtypes (MDA-MB-231: triple negative, MCF-7: ER/PR positive) were selected and exposed to various concentrations of riproximin. Transcriptome and proteomic data revealed that riproximin can up-regulate the three ACGs in breast cancer cells (Fig. 3). Comparatively, more prominent induction of the three ACGs was observed in MCF-7 (up to 3.3fold NOXA, 8.9fold PAR-4, 9fold TRAIL) as compared to MDA-MB-231 cells (up to 3.8fold NOXA, 2.8fold PAR-4, 1.5fold TRAIL). In a previous study, we reported that riproximin can induce significant cytotoxic effects in MCF-7 cells after 48 hours of exposure at much lower concentrations (IC50:0.38ng/ml) as compared to MDA-MB-231 (IC50:3.6ng/ml) cells [26]. Based on previous data and current findings, it can be concluded that breast cancer cells with particular molecular sub-type (e.g., ER/PR positive status) are more sensitive towards riproximin exposure. One potential explanation could be that riproximin interacts with the cells through its lectin binding domains (B-chain) preferably to cell surface glycans (NA2/NA3) and N-acetyl-D-galactosamine (GalNAc/Tn Antigen) [20]. Thus, the presence and saturation status of specific glyco-targets along with the potential involvement of other cell surface receptors can modify the subsequent binding capacity and internalization of riproximin. Other than this, the possibility of wide-ranged downstream signalling cascades being targeted by riproximin in a cell-specific manner cannot be ruled out. As far as riproximin mediated induction of ACGs is concerned, the phenomenon is not restricted to breast cancer cells only, as we recently witnessed that this plant protein can induce the ACGs in multiple colorectal cancer cell lines [42]. To summarize, riproximin mediated up-regulation of ACGs can be exploited against various malignancies provided with further detailed pre-clinical investigations.
Bioinformatics is an emerging interdisciplinary field being exploited in modern day biology to understand complex cellular mechanisms including roles of potential genes/proteins within a cell and extrapolation of the wet-lab results. With this notion, we were interested to highlight the molecular significance of ACGs in cell environment by using various in silico approaches. In the PPI based clustering built with the help of STRING and Cytoscape software, the ACGs showed independent clusters, which in turn, reflects their minimal interdependency on each other (Fig. 4A). If so, this fact is of particular importance as it shows up-regulation of the ACGs via riproximin can initiate distinct and independent cascades to induce antineoplastic effects via multiple molecular directions. TP53 (a master regulator of apoptosis) and DIABLO (a proapoptotic mitochondrial protein) were the only two genes connected between NOXA and TRAIL clusters. The substantial involvement of NOXA and TRAIL in apoptotic routes is a known fact, thus linking bridges between these two ACGs via TP53 and DIABLO is not a surprising observation. PPI networks of the ACGs also showed presence of key enzymes, transcription factors and kinases in the clusters, which means ACGs are involved in mechanistic routes responsible for enzymatic activities, gene expression regulations and phosphorylation steps, hence bear a wide-ranged responsibility inside cells. GO enrichment analysis revealed a huge number of BP (440), MF (28), CC (14) and KEGG pathways (71) being associated with the ACGs (Fig. 4B-E). Mainly these CC, MF and BP include GTPase complex formation, their corresponding activities and apoptotic signalling cascades, respectively. KEGG pathways data was also very enriched where ACGs were shown to be involved in vital mechanisms including survival (PI3K/Akt), apoptosis/necroptosis (caspases and BCL family), chemokine networking, TNF and RAS/RAF signalling cascades. All in all, the bioinformatic approach showed that the ACGs are extremely crucial factors involved in multidirectional aspects of a cell life and need due attention to explore their corresponding prognostic/therapeutic relevance in cancers.
Overall, the findings of this study reflected a distinct expression pattern of ACGs in breast cancer clinical isolates. Targeting these endogenous enemies of transformed cells can be instrumental in the field of cancer therapeutics. Along with synthetic approaches being tested to induce ACGs in cancer cells, naturally occurring compounds with a safe biological profile can be alternative options. In this context, riproximin with its already known substantial antineoplastic effects can be a lead compound for further development. However, further pre-clinical investigations are needed to understand events being operated by riproximin to induce ACGs.