Alessandro Bombonati

BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment: Implications for breast cancer prevention with antioxidant therapies

Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary breast cancer. Similarly, downregulation of BRCA1 protein expression is observed in the majority of basal-like breast cancers. Here, we set out to study the effects of BRCA1 mutations on oxidative stress in the tumor microenvironment. To mimic the breast tumor microenvironment, we utilized an in vitro co-culture model of human BRCA1-mutated HCC1937 breast cancer cells and hTERT-immortalized human fibroblasts. Notably, HCC1937 cells induce the generation of hydrogen peroxide in the fibroblast compartment during co-culture, which can be inhibited by genetic complementation with the wild-type BRCA1 gene. Importantly, treatment with powerful antioxidants, such as NAC and Tempol, induces apoptosis in HCC1937 cells, suggesting that microenvironmental oxidative stress supports cancer cell survival. In addition, Tempol treatment increases the apoptotic rates of MDA-MB-231 cells, which have wild-type BRCA1, but share a basal-like breast cancer phenotype with HCC1937 cells. MCT4 is the main exporter of L-lactate out of cells and is a marker for oxidative stress and glycolytic metabolism. Co-culture with HCC1937 cells dramatically induces MCT4 protein expression in fibroblasts, and this can be prevented by either BRCA1 overexpression or by pharmacological treatment with NAC. We next evaluated caveolin-1 (Cav-1) expression in stromal fibroblasts. Loss of Cav-1 is a marker of the cancer-associated fibroblast (CAF) phenotype, which is linked to high stromal glycolysis, and is associated with a poor prognosis in numerous types of human cancers, including breast cancers. Remarkably, HCC1937 cells induce a loss of Cav-1 in adjacent stromal cells during co-culture. Conversely, Cav-1 expression in fibroblasts can be rescued by administration of NAC or by overexpression of BRCA1 in HCC1937 cells. Notably, BRCA1-deficient human breast cancer samples (9 out of 10) also showed a glycolytic stromal phenotype, with intense mitochondrial staining specifically in BRCA1-deficient breast cancer cells. In summary, loss of BRCA1 function leads to hydrogen peroxide generation in both epithelial breast cancer cells and neighboring stromal fibroblasts, and promotes the onset of a reactive glycolytic stroma, with increased MCT4 and decreased Cav-1 expression. Importantly, these metabolic changes can be reversed by antioxidants, which potently induce cancer cell death. Thus, antioxidant therapy appears to be synthetically lethal with a BRCA1-deficiency in breast cancer cells and should be considered for future cancer prevention trials. In this regard, immunostaining with Cav-1 and MCT4 could be used as cost-effective biomarkers to monitor the response to antioxidant therapy.

Oncogenes and inflammation rewire host energy metabolism in the tumor microenvironment: RAS and NFκB target stromal MCT4

Here, we developed a model system to evaluate the metabolic effects of oncogene(s) on the host microenvironment. A matched set of “normal” and oncogenically transformed epithelial cell lines were co-cultured with human fibroblasts, to determine the “bystander” effects of oncogenes on stromal cells. ROS production and glucose uptake were measured by FACS analysis. In addition, expression of a panel of metabolic protein biomarkers (Caveolin-1, MCT1, and MCT4) was analyzed in parallel. Interestingly, oncogene activation in cancer cells was sufficient to induce the metabolic reprogramming of cancer-associated fibroblasts toward glycolysis, via oxidative stress. Evidence for “metabolic symbiosis” between oxidative cancer cells and glycolytic fibroblasts was provided by MCT1/4 immunostaining. As such, oncogenes drive the establishment of a stromal-epithelial “lactate-shuttle”, to fuel the anabolic growth of cancer cells. Similar results were obtained with two divergent oncogenes (RAS and NFκB), indicating that ROS production and inflammation metabolically converge on the tumor stroma, driving glycolysis and upregulation of MCT4. These findings make stromal MCT4 an attractive target for new drug discovery, as MCT4 is a shared endpoint for the metabolic effects of many oncogenic stimuli. Thus, diverse oncogenes stimulate a common metabolic response in the tumor stroma. Conversely, we also show that fibroblasts protect cancer cells against oncogenic stress and senescence by reducing ROS production in tumor cells. Ras-transformed cells were also able to metabolically reprogram normal adjacent epithelia, indicating that cancer cells can use either fibroblasts or epithelial cells as “partners” for metabolic symbiosis. The antioxidant N-acetyl-cysteine (NAC) selectively halted mitochondrial biogenesis in Ras-transformed cells, but not in normal epithelia. NAC also blocked stromal induction of MCT4, indicating that NAC effectively functions as an “MCT4 inhibitor”. Taken together, our data provide new strategies for achieving more effective anticancer therapy. We conclude that oncogenes enable cancer cells to behave as selfish “metabolic parasites”, like foreign organisms (bacteria, fungi, viruses). Thus, we should consider treating cancer like an infectious disease, with new classes of metabolically targeted “antibiotics” to selectively starve cancer cells. Our results provide new support for the “seed and soil” hypothesis, which was first proposed in 1889 by the English surgeon, Stephen Paget.

Cytokeratin profiles of male breast cancers

Aims:  The prognostic factors and expression of molecular markers in male breast carcinomas are similar to those in female breast cancers. The identification of distinct cytokeratin (CK) profiles (basal as opposed to luminal cells) helps to identify subsets of tumours with different clinical behaviour. The aim of this study was to investigate CK expression in male breast cancer.

Lineage-specific T-cell responses to cancer mucosa antigen oppose systemic metastases without mucosal inflammatory disease.

Cancer mucosa antigens are emerging as a new category of self-antigens expressed normally in immunologically privileged mucosal compartments and universally by their derivative tumors. These antigens leverage the established immunologic partitioning of systemic and mucosal compartments, limiting tolerance opposing systemic antitumor efficacy. An unresolved issue surrounding self-antigens as immunotherapeutic targets is autoimmunity following systemic immunization. In the context of cancer mucosa antigens, immune effectors to self-antigens risk amplifying mucosal inflammatory disease promoting carcinogenesis. Here, we examined the relationship between immunotherapy for systemic colon cancer metastases targeting the intestinal cancer mucosa antigen guanylyl cyclase C (GCC) and its effect on inflammatory bowel disease and carcinogenesis in mice. Immunization with GCC-expressing viral vectors opposed nascent tumor growth in mouse models of pulmonary metastasis, reflecting systemic lineage-specific tolerance characterized by CD8(+), but not CD4(+), T-cell or antibody responses. Responses protecting against systemic metastases spared intestinal epithelium from autoimmunity, and systemic GCC immunity did not amplify chemically induced inflammatory bowel disease. Moreover, GCC immunization failed to promote intestinal carcinogenesis induced by germ-line mutations or chronic inflammation. The established role of CD8(+) T cells in antitumor efficacy, but CD4(+) T cells in autoimmunity, suggests that lineage-specific responses to GCC are particularly advantageous to protect against systemic metastases without mucosal inflammation. These observations support the utility of GCC-targeted immunotherapy in patients at risk for systemic metastases, including those with inflammatory bowel disease, hereditary colorectal cancer syndromes, and sporadic colorectal cancer.

Phosphorylation of Vasodilator-Stimulated Phosphoprotein Ser239 Suppresses Filopodia and Invadopodia in Colon Cancer

In colorectal cancer, the antitumorigenic guanylyl cyclase C (GCC) signalome is defective reflecting ligand deprivation from downregulation of endogenous hormone expression. Although the proximal intracellular mediators of that signal transduction system, including cyclic guanosine monophosphate (cGMP) and cGMP‐dependent protein kinase (PKG), are well characterized, the functional significance of its distal effectors remain vague. Dysregulation of ligand‐dependent GCC signaling through vasodilator‐stimulated phosphoprotein (VASP), an actin‐binding protein implicated in membrane protrusion dynamics, drastically reduced cGMP‐dependent VASP phosphorylation levels in colorectal tumors from patients. Restoration of cGMP‐dependent VASP phosphorylation by GCC agonists suppressed the number and length of locomotory (filopodia) and invasive (invadopodia) actin‐based organelles in human colon cancer cells. Membrane organelle disassembly reflected specific phosphorylation of VASP Ser239, the cGMP/PKG preferred site, and rapid VASP removal from tumor cell protrusions. Importantly, VASP Ser239 phosphorylation inhibited the proteolytic function of invadopodia, reflected by suppression of the cancer cell ability to digest DQ‐collagen IV embedded in Matrigel. These results demonstrate a previously unrecognized role for VASP Ser239 phosphorylation, a single intracellular biochemical reaction, as an effective mechanism which opposes tumor cell shape promoting colon cancer invasion and metastasis. Reconstitution of physiological cGMP circuitry through VASP, in turn, represents an attractive targeted approach for patients with colorectal cancer.

Ductal carcinoma in situ of the breast: the importance of morphologic and molecular interactions

Ductal carcinoma in situ (DCIS) of the breast is a lesion characterized by significant heterogeneity, in terms of morphology, immunohistochemical staining, molecular signatures, and clinical expression. For some patients, surgical excision provides adequate treatment, but a subset of patients will experience recurrence of DCIS or progression to invasive ductal carcinoma (IDC). Recent years have seen extensive research aimed at identifying the molecular events that characterize the transition from normal epithelium to DCIS and IDC. Tumor epithelial cells, myoepithelial cells, and stromal cells undergo alterations in gene expression, which are most important in the early stages of breast carcinogenesis. Epigenetic modifications, such as DNA methylation, together with microRNA alterations, play a major role in these genetic events. In addition, tumor proliferation and invasion is facilitated by the lesional microenvironment, which includes stromal fibroblasts and macrophages that secrete growth factors and angiogenesis-promoting substances. Characterization of DCIS on a molecular level may better account for the heterogeneity of these lesions and how this manifests as differences in patient outcome and response to therapy. Molecular assays originally developed for assessing likelihood of recurrence in IDC are recently being applied to DCIS, with promising results. In the future, the classification of DCIS will likely incorporate molecular findings along with histologic and immunohistochemical features, allowing for personalized prognostic information and therapeutic options for patients with DCIS. This review summarizes current data regarding the molecular characterization of DCIS and discusses the potential clinical relevance.

Guanylyl cyclase C is a specific marker for differentiating primary and metastatic ovarian mucinous neoplasms

Aims: The aim was to assess the value of GCC in distinguishing primary ovarian mucinous neoplasms from metastatic mucinous adenocarcinomas with ovarian involvement. Guanylyl cyclase C (GCC) is a brush border membrane receptor for the endogenous peptides guanylin and uroguanylin, and the homologous diarrhoeagenic bacterial heat‐stable enterotoxins that is selectively expressed by epithelial cells from the duodenum to the rectum, but not by normal epithelia of the stomach or oesophagus, or normal extramucosal cells in humans.

Metastases to and from the Breast

Breast cancer is a common source of systemic metastatic disease. Distinguishing metastatic breast cancer from other types of malignancies can be diagnostically challenging but is important for correct treatment and prognosis. Nonmammary tumors can also metastasize to the breast, although this is a rare phenomenon. Differentiating a metastasis to the breast from a primary breast cancer can likewise be difficult. Knowledge of the clinical history and careful morphologic evaluation are the cornerstones of diagnosis. A panel of immunohistochemical stains tailored to the differential diagnosis at hand can provide helpful information in ambiguous cases.

Abstract 5805: Myosin 1e colocalization with β1-intergin and association with tumor progression in colorectal cancer

For colorectal cancer (CRC), the risk of relapse among stage II and stage III a/b patients is 20-30% and the benefit of treating these patients with chemotherapy is uncertain. As a result, many patients are undertreated, putting them at increased risk for disease relapse, or overtreated, exposing them to unnecessary and harmful chemotherapy with little potential benefit. Therefore, defining prognostic biomarkers to more accurately determine each person’s risk of relapse and need for chemotherapy is a priority in CRC research. Promising targets for biomarker discovery are actin-binding proteins. As a broad class, these proteins regulate the actin cytoskeleton and serve as direct, proximal regulators of invasive and metastatic phenotypes. Further, the prognostic utility of several of these proteins has recently been reported in the literature. Myosin 1e (Myo1e) a long-tailed, class I myosin, is one such protein with proposed clinical utility as a prognostic biomarker. In kidney podocyte cells, Myo1e regulates endocytosis, adhesion, migration, and invadosome dynamics. Despite its expression in numerous cancers, the functional role of Myo1e specifically in cancer cells and its association with tumor progression remains elusive. To define a role in cancer cells, the localization of Myo1e was examined in vitro by immunofluorescence in T84 human CRC cells. Myo1e co-localized with actin, cortactin and β1 integrin at membrane ruffles, which regulate integrin endocytosis and trafficking. Interestingly, both Myo1e and β1 integrin also co-localized with caveolin-1, a regulator of integrin endocytosis, suggesting Myo1e expression may regulate caveolae-dependent integrin endocytosis and trafficking. To evaluate potential prognostic utility, Myo1e expression in colorectal tumors and matched normal adjacent tissue (NAT) was examined by immunohistochemistry in a tissue microarray constructed from duplicate tissue cores from 119 CRC patients. The patient cohort was well balanced across TNM stage: stage 0 (10.1%), stage 1 (20.2%), stage 2 (26.9%), stage 3 (31.1%), and stage 4 (12.6%), and the majority (68.9%) of CRC tissues examined were of moderate tumor grade. The ratio of Myo1e expression in tumors compared to NAT was significantly correlated with clinicopathologic indicators of disease progression, including clinical stage, depth of invasion at the primary tumor (T-score), and lymph node metastasis. Together, these data suggest Myo1e expression correlates with tumor progression and may regulate invasive phenotypes through integrin trafficking pathways. Examining the prognostic utility and biologic function of Myo1e may a define a novel biomarker with translational utility for improved clinical management of colorectal cancer patients. Citation Format: Jeffrey Pfannenstein, Filippo Bori, Alessandro Bombonati, David Zuzga. Myosin 1e colocalization with β1-intergin and association with tumor progression in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5805. doi:10.1158/1538-7445.AM2017-5805