Molecular profiling of microinvasive breast cancer microenvironment progression

Tumors develop by progression through a series of stages. It is now widely accepted that cancer is attributed to the accumulation of genetic alterations in cells. Every cell of the tumor microenvironment is constantly changing in the flow of the cancer progression. The possible role of the tumor microenvironment in neoplastic development has been investigated since the late nineteenth century, with studies published by Stefano Paget in 1989 [1]. The structure and functions of the tumor microenvironment, as well as the relationships with the neoplasia, allow to define more precise prognostic and therapeutic directions.

Breast cancer carcinogenesis is well known, characterized by well defined stages, starting from the atypical ductal hyperplasia progressing to ductal carcinoma in situ (DCIS) and ending, although not necessarily, with the invasive breast cancer (IBC). [2].

In breast cancer, epithelial cells require the stroma to meet their needs of nutrition, waste removal, and structure. It has become clear in recent years that stroma is, indeed, essential for tumor maintenance and growth which can also provide protection from the human immune system attacking the cancer cells [3, 4]. The tumor microenvironment is characterized by an increased number of fibroblasts, expressing alpha-smooth muscle actin, so-called cancer associated fibroblasts (CAFs). Therefore it is important to integrate gene expression changes of both tumoral cells and cancer-associated stroma, occurring during the difference phases of tumor progression. For this reason we focused our attention on a specific kind of breast cancer such as the microinvasive breast carcinoma (MIBC), which is a rare entity in which an invasive component not exceeding 1 mm is found, mostly in a DCIS setting [5]. MIBC accounts for about 5–10% of DCIS with a very good overall prognosis for the patients [6]. The peculiar characteristic of this tumor histotype, that meets our needs, is that we are able to identify on the same tumor section at the meantime all phases of breast cancer progression: normal tissue, DCIS and invasive foci with the respective surrounding stroma.

Formalin-fixed, paraffin-embedded (FFPE) tissue samples stored in diagnostic pathology archives represent an invaluable bio-bank for retrospective clinical research. This interest is primarily driven by the fact that the process of creating FFPE tissue is the most common technique used by clinical and/or research pathologists for tissue processing, evaluation, diagnostics, immunoanalysis, preservation, and archiviation. The use of FFPE samples in molecular studies presents some great advantages, for example, these types of samples are available and readily accessible in vast quantities, which is a very important element considering a rare disease such as MIBC. The cost associated with their storage is low, as well, and the significant association between pathological and clinical annotations makes FFPE tissue an attractive specimen for biomarker discovery. In particular, thanks to the use of FFPE histological sections, a much higher resolution level is reached, which allows an accurate distinction of tumor areas with specific characteristics that otherwise would not be identifiable.

The aim of the present study is to analyze in MIBC the transcriptome of mammary neoplastic epithelium at different stages of progression together with the respective stroma in order to obtain an overview of the temporal modulation of the gene expression profile during tumor progression enriched by the gene expression profile of the stroma surrounding each tumoral portion at each stage.

Breast cancer is the most common malignancy and the leading cause of cancer-related death in women worldwide. The microenvironment of these cancers is now recognized as a critical participant in tumor progression. Recent data demonstrate significant gene expression in cells composing the microenvironment during disease progression, which can be explored as biomarkers and targets for therapy. Indeed, gene expression signatures derived from tumor stroma have been linked to clinical outcomes. The tumor microenvironment has assumed a progressively increasing importance over the years; infact a continuos interaction is obtained: on one hand, the tumor is able to influence the microenvironment thanks to extracellular signals, promoting phenomena such as neoangiogenesis and immuno-tolerance; on the other, the cells of the microenvironment favor tumor progression. There is increasing interest in refining our current understanding of the tumor microenvironment. An in-depth study of the tumor microenvironment, can provide information on both the molecular mechanisms underlying the progression as well as on possible etiological factors. In fact, except for some hypotheses of viral etiology [13], we are not yet aware of the etiological cause of breast cancer.

The aim of this study was to analyze the gene expression pattern of microdissected tumoral epithelial cell areas related to each phase of tumoral progression in breast cancer (CIS, EIF and IBC) compared to the normal epithelial cells area (NBE). At the same time we studied also the stromal portions around the tumoral epithelial areas (S-EIF and S-IBC) in comparison to the stromal area surrounding the normal epithelial mammary tissue (S-NBE). We decided to collect these areas, respectively, from 7 patients utilizing the MIBC type, in which cancer progression phases are still very distinguishable. This approach has the advantage of giving a more integral view of the transcriptome changes occurring during cancer progression and allows the investigation of interactions between compartments. The approach can also give insights on the molecular mechanisms that govern cell–cell interactions.

From all gene expression level comparisons, some key aspects have emerged. Focusing on the overall DEGs in epithelial portions, the main pathways in which DEGs were grouped are the endocytosis process, the pathways in cancer and interestingly the fructose and mannose metabolism. Cancer metabolism is essential for the maintenance of cell proliferation in a tumor. The pioneering studies of O. Warburg [14] asserted that a cancer cell needs an increase in glycolysis and a decrease of oxidative metabolism. Nowadays, after several further investigations, the starting concept has been revisited. Metabolism heterogeneity is well known in cancer, both for cancer cells and for the cells of the microenvironment. So a single metabolic program can not be representative of the global metabolism of a tumor. Infact, fructose metabolism, for instance, is different from that of glucose. Through the PPP, fructose induces NADPH and nucleotides synthesis. Besides, glucose also generates fructose through a specific pathway, the polyol pathway, and some of its metabolites (ex. glycolaldehyde and glyoxal) can affect cell survival [15]. Through this mechanism, this type of metabolism can have a role in neoplastic growth.

The overall DEGs identified in stromal samples, are grouped essentially into the focal adhesion process, the extracellular matrix (ECM) receptor interaction pathway and regulation of actin cytoskeleton pathway. All these processes are linked to cell motility, essential for invasion and for metastasis formation. Cancer cell movement during invasion is a complex system made mainly of membrane protrusions (lamellipodia) arising at the leading edge of migrating cancer cells after activation by extracellular stimuli. Afterwards, the leading membrane is fixed by nascent sites of attachment (focal adhesions) [16]. The F-actin stress fibres contract, creating the tension needed to drag the cell forward, with loss of adhesion at the rear of the cell, so the cell retracts and is dragged in the direction of migration [17]. The involvement, that we detected, of these pathways in the stroma-derived samples is, therefore, perfectly in agreement with the literature, since in our study tumor epithelial cells are progressing towards a real invasion supported by the stromal cells in the process of tumoral progression.

Among all DEGs derived from the comparisons done within tumoral epithelial samples, we identified some key genes that gradually decrease or increase their expression with tumoral progression: KRT15, SFRP1 and MARS. KRT15 (Cytokeratin 15) is a cytoskeletal protein, expressed essentially in the epithelial cells and considered a marker of epithelial stem cells [18]. In our samples we observed a significant decrease of expression in tumoral cells compared to the normal tissue, this is in accordance with Shen et al. in a study on esophageal squamous carcinoma [19] even if in literature there are conflicting results about its role in cancer [20‐22]. SFRP1 (Secreted Frizzled Related Protein 1) is a member of SFRP family whose function is to modulate Wnt signaling through direct interaction with Wnts. This gene has already been found involved in breast cancer tumor progression as a tumor suppressor gene and moreover it has been proposed as a target gene for early diagnosis [23]. In our samples SFRP1 expression levels are in complete accordance with the literature, with a decrease during tumoral progression [23]. MARS (Methionyl-TRNA Synthetase) is a member of the class I family of aminoacyl-tRNA synthetases. We found this gene with a gradual increase of expression in tumoral portions, in accordance with the paper of Kim et al. [24] that observed MARS overexpression in non-small cell lung cancer, associated also with a poor prognosis.

The key genes identified among DEGs obtained from the comparisons within the stromal portions are: STAT2, NFE2L1, SIN3B and NOTCH2. All these genes showed a gradual upregulation during the tumoral progression. STAT2 (signal transducer and activator of transcription 2) is a member of STAT family proteins generally involved in response to interferon. In particular STAT2 is a necessary transcription factor in the IFN-α/β signaling pathway [25]. Ogony et al. [26] studied STAT2 in breast cancer cells as a key regulator of the expression of IFITM1 (interferon-induced transmembrane protein 1); together they are involved in the IFNα signaling pathway, in particular their overexpression promote cancer aggressiveness in breast cancer, that agrees with our data. Moreover in literature some papers are already reporting data about IFNα immunotherapy and STAT2 status in melanoma [27] and in other type of diseases [28]. NFE2L1 (nuclear factor, erythroid 2 like 1) is a protein that is involved in globin gene expression in erythrocytes, this protein is not yet well studied, the most important function seems to be related to proteasome process [29]. Very different it is the case of SIN3B (SIN3 transcription regulator family member B), a well known protein that interacts with MYC (MYC proto-oncogene, BHLH transcription factor), which was observed promoting cancer progression and metastasis in breast cancer [30] in accordance with our data. Also NOTCH2 (neurogenic locus notch homolog protein 2) is a very well known protein, that functions as a receptor for membrane-bound ligands jagged-1 (JAG1), jagged-2 (JAG2) and delta-1 (DLL1) to regulate cell-fate determination. Several studies have been conducted on NOTCH2 and cancer, not all in accordance with our results. Some studies describe NOTCH2 as a tumor suppressor gene in breast cancer [31, 32] while as an oncogene in bladder cancer [33] promoting cancer growth and metastasis through epithelial–mesenchymal transition (EMT), which process is fully consistent with our findings. It is important to point out that this is the first time that STAT2, NFE2L1, SIN3B and NOTCH2 genes are described associated to the cancer stroma.

Because of the heterogeneity of cancer cells, each tumor differs in its metabolic status [34]. This is well demonstrated in our samples. In detail, we can deduce that the CIS is a so-called oxidative tumor, because no glycolysis is activated, but there is a great activation of TCA cycle from which the cell receives the energy. This is in accordance with some studies demonstrating that there are tumors, such as the oxidative tumors, where glycolysis is not predominant [35]. Furthermore, we observed in the CIS compartment, a release of lactate from the cell due to an upregulation of the MCT4 gene. We can assume, therefore, that the lactate, released by CIS, enters in EIF cells, which present an upregulation of the MCT1 gene. EIF cells, which are about to invade, like CIS cells, have a lower activation of glycolysis in favor of the TCA cycle. Also these tumoral cells behave like oxidative tumor cells. Moreover, in the EIF cells, a release of GLN is detected, which enters in the surrounding S-EIF cells and is used as energy fuel, generating GLU through the glutaminolysis. According to our observations, the S-EIF compartment undergo aerobic glycolysis and generate high levels of fuels like fatty acids, lactate, ketonic bodies in compliance to what the reverse Warburg effect describes. It is well known, infact, that in the reverse Warburg effect, CAFs “feed” the tumoral cells with glycolysis and fatty acid and ketonic bodies synthesis [36, 37]. In turn, cancer cells produce ATP through the TCA cycle and mitochondrial oxidative phosphorylation system (OXPHOS) [38, 39], as we observed in EIF cells. When we focus on the invasion process, the IBC cells show a similar reverse Warburg metabolic situation as in the EIF cells. S-IBC cells are, indeed, characterized by a glycolytic metabolism with release of lactate that enters the IBC tumoral cells, which show an oxidative metabolism.

Our data describe, by the use of LCM on FFPE tissues, the changes of gene expression values during cancer progression in the epithelial cells enriched by the gene expression changes of the surrounding stromal cells. This is the first time that such gene expression values are obtained from FFPE microdissected areas localized on the same tissue section. It is well known that CAFs are necessary for the development and the maintenance of the tumor and particularly for tumor progression. Since we are facing a new phase where there is a conversion from a cancer cell-centric strategy to a stroma-centric strategy, it is crucial to pursue further investigations to better clarify the role of CAFs in tumor progression.