Blocking soluble TNFα sensitizes HER2-positive breast cancer to trastuzumab through MUC4 downregulation and subverts immunosuppression.

BACKGROUND: The success of HER2-positive (HER2+) breast cancer treatment with trastuzumab, an antibody that targets HER2, relies on immune response. We demonstrated that TNFα induces mucin 4 (MUC4) expression, which shields the trastuzumab epitope on the HER2 molecule decreasing its therapeutic effect. Here, we used mouse models and samples from HER2+ breast cancer patients to unravel MUC4 participation in hindering trastuzumab effect by fostering immune evasion. METHODS: We used a dominant negative TNFα inhibitor (DN) selective for soluble TNFα (sTNFα) together with trastuzumab. Preclinical experiments were performed using two models of conditionally MUC4-silenced tumors to characterize the immune cell infiltration. A cohort of 91 patients treated with trastuzumab was used to correlate tumor MUC4 with tumor-infiltrating lymphocytes. RESULTS: In mice bearing de novo trastuzumab-resistant HER2+ breast tumors, neutralizing sTNFα with DN induced MUC4 downregulation. Using the conditionally MUC4-silenced tumor models, the antitumor effect of trastuzumab was reinstated and the addition of TNFα-blocking agents did not further decrease tumor burden. DN administration with trastuzumab modifies the immunosuppressive tumor milieu through M1-like phenotype macrophage polarization and NK cells degranulation. Depletion experiments revealed a cross-talk between macrophages and NK cells necessary for trastuzumab antitumor effect. In addition, tumor cells treated with DN are more susceptible to trastuzumab-dependent cellular phagocytosis. Finally, MUC4 expression in HER2+ breast cancer is associated with immune desert tumors. CONCLUSIONS: These findings provide rationale to pursue sTNFα blockade combined with trastuzumab or trastuzumab drug conjugates for MUC4+ and HER2+ breast cancer patients to overcome trastuzumab resistance.

Proteoglycans: A Tool for Detecting Hyaluronan by ELISA-Like Methods.

Hyaluronan is a non-sulfated glycosaminoglycan synthesized on the plasma membrane of almost all mammalian cells, which can interact with different proteoglycans of the extracellular matrix. Aggrecan, versican, neurocan, and brevican are proteoglycans whose structures present a specific protein domain called "link module," which allows hyaluronan binding. Therefore, they can be helpful for assays that detect hyaluronan. For example, ELISA-like methods developed to measure hyaluronan amounts in solution are based on specific interactions between this molecule and the link module present in aggrecan or other hyaluronan-binding proteins (hyaladherins).

Halting ErbB-2 isoforms retrograde transport to the nucleus as a new theragnostic approach for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is clinically defined by the absence of estrogen and progesterone receptors and the lack of membrane overexpression or gene amplification of receptor tyrosine kinase ErbB-2/HER2. Due to TNBC heterogeneity, clinical biomarkers and targeted therapies for this disease remain elusive. We demonstrated that ErbB-2 is localized in the nucleus (NErbB-2) of TNBC cells and primary tumors, from where it drives growth. We also discovered that TNBC expresses both wild-type ErbB-2 (WTErbB-2) and alternative ErbB-2 isoform c (ErbB-2c). Here, we revealed that the inhibitors of the retrograde transport Retro-2 and its cyclic derivative Retro-2.1 evict both WTErbB-2 and ErbB-2c from the nucleus of BC cells and tumors. Using BC cells from several molecular subtypes, as well as normal breast cells, we demonstrated that Retro-2 specifically blocks proliferation of BC cells expressing NErbB-2. Importantly, Retro-2 eviction of both ErbB-2 isoforms from the nucleus resulted in a striking growth abrogation in multiple TNBC preclinical models, including tumor explants and xenografts. Our mechanistic studies in TNBC cells revealed that Retro-2 induces a differential accumulation of WTErbB-2 at the early endosomes and the plasma membrane, and of ErbB-2c at the Golgi, shedding new light both on Retro-2 action on endogenous protein cargoes undergoing retrograde transport, and on the biology of ErbB-2 splicing variants. In addition, we revealed that the presence of a functional signal peptide and a nuclear export signal (NES), both located at the N-terminus of WTErbB-2, and absent in ErbB-2c, accounts for the differential subcellular distribution of ErbB-2 isoforms upon Retro-2 treatment. Our present discoveries provide evidence for the rational repurposing of Retro-2 as a novel therapeutic agent for TNBC.

Canonical ErbB-2 isoform and ErbB-2 variant c located in the nucleus drive triple negative breast cancer growth.

Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2/HER2, a receptor tyrosine kinase. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Clinical biomarkers and targeted therapies for this disease remain elusive, so chemotherapy has been the standard of care for early and metastatic TNBC. Our present findings placed ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing events, using a PCR-sequencing approach combined with an RNA interference strategy, revealed that TNBC cells express either the canonical (wild-type) ErbB-2, encoded by transcript variant 1, or the non-canonical ErbB-2 isoform c, encoded by alternative variant 3 (RefSeq), or both. These ErbB-2 isoforms function in the nucleus as transcription factors. Evicting both from the nucleus or silencing isoform c only, blocks TN cell and tumor growth. This reveals not only NErbB-2 canonical and alternative isoforms role as targets of therapy in TNBC, but also isoform c dominant oncogenic potential. Furthermore, we validated our findings in the clinic and observed that NErbB-2 correlates with poor prognosis in primary TN tumors, disclosing NErbB-2 as a novel biomarker for TNBC. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type RefSeq proteins, which conserve the canonical domains and are located in their classical cellular compartments.

Nuclear PDCD4 Expression Defines a Subset of Luminal B-Like Breast Cancers with Good Prognosis.

The hormone receptor-positive (estrogen and/or progesterone receptor (PR)-positive) and HER2-negative breast cancer (BC) subtype is a biologically heterogeneous entity that includes luminal A-like (LumA-like) and luminal B-like (LumB-like) subtypes. Decreased PR levels is a distinctive biological feature of LumB-like tumors. These tumors also show reduced sensitivity to endocrine therapies and poorer prognosis than LumA-like tumors. Identification of biomarkers to accurately predict disease relapse in these subtypes is crucial in order to select effective therapies. We identified the tumor suppressor PDCD4 (programmed cell death 4), located in the nucleus (NPDCD4), as an independent prognostic factor of good clinical outcome in LumA-like and LumB-like subtypes. NPDCD4-positive LumB-like tumors presented overall and disease-free survival rates comparable to those of NPDCD4-positive LumA-like tumors, indicating that NPDCD4 improves the outcome of LumB-like patients. In contrast, NPDCD4 loss increased the risk of disease recurrence and death in LumB-like compared with LumA-like tumors. This, along with our results showing that LumB-like tumors present lower NPDCD4 positivity than LumA-like tumors, suggests that NPDCD4 loss contributes to endocrine therapy resistance in LumB-like BCs. We also revealed that PR induces PDCD4 transcription in LumB-like BC, providing a mechanistic explanation to the low PDCD4 levels in LumB-like BCs lacking PR. Finally, PDCD4 silencing enhanced BC cell survival in a patient-derived explant model of LumB-like disease. Our discoveries highlight NPDCD4 as a novel biomarker in LumA- and LumB-like subtypes, which could be included in the panel of immunohistochemical markers used in the clinic to accurately predict the prognosis of LumB-like tumors.

Nuclear ErbB-2: a Novel Therapeutic Target in ErbB-2-Positive Breast Cancer?

Membrane overexpression of ErbB-2 (MErbB-2), a member of the ErbB family of receptor tyrosine kinases, occurs in 15-20% of breast cancers (BC) and constitutes a therapeutic target in this BC subtype (ErbB-2-positive). Although MErbB-2-targeted therapies have significantly improved patients' clinical outcome, resistance to available drugs is still a major issue in the clinic. Lack of accurate biomarkers for predicting responses to anti-ErbB-2 drugs at the time of diagnosis is also an important unresolved issue. Hence, a better understanding of the ErbB-2 signaling pathway constitutes a critical task in the battle against BC. In its canonical mechanism of action, MErbB-2 activates downstream signaling pathways, which transduce its proliferative effects in BC. The dogma of ErbB-2 mechanism of action has been challenged by the demonstration that MErbB-2 migrates to the nucleus, where it acts as a transcriptional regulator. Accumulating findings demonstrate that nuclear ErbB-2 (NErbB-2) is involved in BC growth and metastasis. Emerging evidence also reveal a role of NErbB-2 in the response to available anti-MErbB-2 agents. Here, we will review NErbB-2 function in BC and will particularly discuss the role of NErbB-2 as a novel target for therapy in ErbB-2-positive BC.

Efectos nucleares del receptor Tirosina Quinasa ErbB-2 en cáncer de mama / NUCLEAR EFFECTS OF THE ErbB-2 RECEPTOR TYROSINE KINASE IN BREAST CANCER

Aproximadamente 15-20% de los cánceres de mama (CM) presentan sobre- expresión en la membrana citoplasmática de ErbB-2 (MErbB-2), un miembro de la familia ErbBs de receptores con actividad de tirosina quinasa, o bien presentan amplificación del gen. Antes del desarrollo de terapias dirigidas contra el MErbB-2, este subtipo de CM, denominado ErbB-2-positivo, estaba asociado con un aumento en el potencial metastásico del tumor y un mal pronóstico. Estas terapias han aumentado significativamente la sobrevida global y el porcentaje de enfermos curados. Sin embargo, la resistencia a las terapias disponibles actualmente es todavía un importante problema en la clínica. Actuando por su mecanismo clásico, el MErbB-2 activa cascadas de señalización que transducen sus efectos en el cáncer de mama. La presencia del ErbB-2 en el núcleo fue descubierta hace más de veinte años. Evidencias experimentales proporcionadas por varios grupos de investigación, incluyendo el nuestro, revelaron una función no canónica del ErbB-2 en el núcleo celular donde actúa como un regulador de transcripción. Nuestros hallazgos demostraron que el ErbB-2 nuclear estimula el crecimiento del CM, el desarrollo de metástasis y la resistencia a las terapias utilizadas actualmente

Membrane overexpression of ErbB-2 (MErbB-2), a member of the ErbBs family of receptor tyrosine kinases, or ErbB-2 gene amplification, occurs in 15-20% of breast cancers (BC). Until the development of MErbB-2-targeted therapies, this BC subtype, called ErbB-2-positive, was associated with increased metastatic potential and poor prognosis. Although the overall survival and cure rates have improved significantly with such therapies, resistance to available drugs is still a major clinical issue. In its classical mechanism, MErbB-2 activates downstream signal cascades, which transduce its effects in BC. The fact that ErbB-2 is also present at the nucleus of BC cells was discovered over twenty years ago. Also, compelling evidence revealed a non-canonical function of nuclear ErbB-2 as a transcriptional regulator. Since deeper understanding of nuclear ErbB-2 actions would be critical to disclose its role as a biomarker and a target of therapy in BC, we will here review its function in BC, focusing on its role in growth, metastatic spreading, and response to currently available MErbB-2 positive BC therapies.

ErbB-2 nuclear function in breast cancer growth, metastasis and resistance to therapy.

Approximately 15-20% of breast cancers (BC) show either membrane overexpression of ErbB-2 (MErbB-2), a member of the ErbBs family of receptor tyrosine kinases, or ERBB2 gene amplification. Until the development of MErbB-2-targeted therapies, this BC subtype, called ErbB-2-positive, was associated with increased metastatic potential and poor prognosis. Although these therapies have significantly improved overall survival and cure rates, resistance to available drugs is still a major clinical issue. In its classical mechanism, MErbB-2 activates downstream signaling cascades, which transduce its effects in BC. The fact that ErbB-2 is also present in the nucleus of BC cells was discovered over twenty years ago. Also, compelling evidence revealed a non-canonical function of nuclear ErbB-2 as a transcriptional regulator. As a deeper understanding of nuclear ErbB-2 actions would be crucial to the disclosure of its role as a biomarker and a target of therapy in BC, we will here review its function in BC, in particular, its role in growth, metastatic spreading and response to currently available MErbB-2-positive BC therapies.

Increased hyaluronan levels and decreased dendritic cell activation are associated with tumor invasion in murine lymphoma cell lines.

Hyaluronan (HA), a component of the extracellular matrix surrounding tumors, modulates tumor progression and the immune response. Dendritic cells (DC) may tolerize or stimulate immunity against cancer. In this report, we study the association between tumor progression, HA levels and DC activation in a lymphoma model. Mice injected with the cells with highest invasive capacity (LBR-) presented increased HA in serum and lymph nodes, and decreased DC activation in infiltrated lymph nodes and liver. These findings could be related to lack of an effective antitumor immune response and suggest that serum HA levels could have a prognostic value in hematological malignancies.

Hyaluronan induces migration of multidrug-resistant lymphoma cell lines in vitro through Tiam1 activation by a PI3K-dependent mechanism.

Hyaluronan (HA) modulates multidrug resistance (MDR) as well as cell migration. Tiam1 is involved in cytoskeleton reorganization during tumor invasion. In this report we show the relationship among HA, Tiam1, migration and MDR in murine lymphoma cell lines. We observed that MDR cells presented higher migratory capacity towards HA in vitro as well as higher constitutive active Tiam1 expression than the sensitive cell line. Besides, HA treatment induced migration towards HA of MDR cell lines through Tiam1 activation by a PI3K-dependent mechanism, showing that disruption of HA signaling would be useful in treatment of MDR hematological malignancies.

PI3K/Akt inhibition modulates multidrug resistance and activates NF-kappaB in murine lymphoma cell lines.

Upregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been described in some tumors related to multidrug resistance (MDR). The aim of this work was to analyze the relationship between PI3K/Akt, MDR and NF-kappaB in murine lymphoma cell lines resistant to vincristine (LBR-V160) and doxorubicin (LBR-D160) as well as in the sensitive line (LBR-). PI3K/Akt activity, analyzed by phosphatidylinositol trisphosphate production and phosphorylated Akt (p-Akt) expression, was higher in the resistant cell lines than in the sensitive one and inhibition with wortmannin or LY294002 improved apoptosis in the resistant cell lines. Vincristine but not doxorubicin increased p-Akt expression whereas co-treatment with PI3K inhibitors and vincristine increased apoptosis in the three cell lines. Wortmannin and LY294002 inhibited P-glycoprotein (Pgp) function and also increased NF-kappaB activity. We concluded that the PI3K/Akt pathway is involved in MDR in lymphoma cell lines and PI3K/Akt inhibition correlates down-regulation of NF-kappaB activity and inhibition Pgp function.

Interaction between dendritic cells and natural killer cells during pregnancy in mice.

A complex regulation of innate and adaptive immune responses at the maternal fetal interface promotes tolerance of trophoblast cells carrying paternally derived antigens. Such regulatory functions involve uterine dendritic cells (uDC) and natural killer (uNK) cells. The existence of a NK and DC "cross talk" has been revealed in various experimental settings; its biological significance ranging from cooperative stimulation to cell lysis. Little is known about the presence or role of NK and DC cross talk at the maternal fetal interface. The present study shows that mouse NK and DC interactions are subject to modulation by trophoblast cells in vitro. This interaction promotes a tolerogenic microenvironment characterized by downregulation of the expression of activation markers on uNK cells and uDC and dominance of Th2 cytokines. NK and DC interactions would also influence uterine cell proliferation and this process would be strongly modulated by trophoblast-derived signals. Indeed; while low proliferation rates were observed upon regular coculture allowing direct contact between uterine cells and trophoblasts, incubation in a transwell culture system markedly increased uterine cell proliferation suggesting that soluble factors are key mediators in the molecular "dialog" between the mother and the conceptus during the establishment of mouse pregnancy. Our data further reveal that the regulatory functions of trophoblast cells associated with tolerance induction are impaired in high abortion murine matings. Interestingly, we observed that secretion of interleukin-12p70 by uDC is dramatically abrogated in the presence of uNK cells. Taken together, our results provide the first evidence that a delicate balance of interactions involving NK cells, DC, and trophoblasts at the mouse maternal fetal interface supports a successful pregnancy outcome.

Hyaluronan oligosaccharides sensitize lymphoma resistant cell lines to vincristine by modulating P-glycoprotein activity and PI3K/Akt pathway.

Multidrug resistance (MDR) is one of the main reasons for failure of cancer therapy. It may be mediated by overexpression of ATP-dependent efflux pumps or by alterations in survival or apoptotic pathways. Fragments generated by enzymatic degradation of hyaluronan (oHA) were able to modulate growth and cell survival and sensitize MDR breast cancer cells to cytotoxic drugs. In this work the relationship between oHA and MDR in lymphoid malignancies was analyzed using murine lymphoma cell lines resistant to doxorubicin (LBR-D160) or vincristine (LBR-V160) and a sensitive line (LBR-). After oHA treatment, higher apoptosis levels were observed in the resistant cell lines than in the sensitive one. Besides, oHA sensitized LBR-D160 and LBR-V160 to vincristine showing increased apoptosis induction when used in combination with vincristine. Native hyaluronan failed to increase apoptosis levels. As different survival factors could be modulated by hyaluronan, we investigated the PI3K/Akt pathway through PIP3 production and phosphorylated Akt (p-Akt) and survivin expression was also evaluated. Our results showed that oHA decreased p-Akt in the 3 cell lines while anti-CD44 treatment abolished this effect. Besides, survivin was downregulated only in LBR-V160 by oHA. When Pgp function was evaluated, we observed that oHA were able to inhibit Pgp efflux in murine and human resistant cell lines in a CD44-dependent way. In summary, we report for the first time that oHA per se modulate MDR in lymphoma cells by decreasing p-Akt as well as Pgp activity, thus suggesting that oHA could be useful in combination with classical chemotherapy in MDR hematological malignancies.