MiR-16 mediates trastuzumab and lapatinib response in ErbB-2-positive breast and gastric cancer via its novel targets CCNJ and FUBP1.

ErbB-2 amplification/overexpression accounts for an aggressive breast cancer (BC) subtype (ErbB-2-positive). Enhanced ErbB-2 expression was also found in gastric cancer (GC) and has been correlated with poor clinical outcome. The ErbB-2-targeted therapies trastuzumab (TZ), a monoclonal antibody, and lapatinib, a tyrosine kinase inhibitor, have proved highly beneficial. However, resistance to such therapies remains a major clinical challenge. We here revealed a novel mechanism underlying the antiproliferative effects of both agents in ErbB-2-positive BC and GC. TZ and lapatinib ability to block extracellular signal-regulated kinases 1/2 and phosphatidylinositol-3 kinase (PI3K)/AKT in sensitive cells inhibits c-Myc activation, which results in upregulation of miR-16. Forced expression of miR-16 inhibited in vitro proliferation in BC and GC cells, both sensitive and resistant to TZ and lapatinib, as well as in a preclinical BC model resistant to these agents. This reveals miR-16 role as tumor suppressor in ErbB-2-positive BC and GC. Using genome-wide expression studies and miRNA target prediction algorithms, we identified cyclin J and far upstream element-binding protein 1 (FUBP1) as novel miR-16 targets, which mediate miR-16 antiproliferative effects. Supporting the clinical relevance of our results, we found that high levels of miR-16 and low or null FUBP1 expression correlate with TZ response in ErbB-2-positive primary BCs. These findings highlight a potential role of miR-16 and FUBP1 as biomarkers of sensitivity to TZ therapy. Furthermore, we revealed miR-16 as an innovative therapeutic agent for TZ- and lapatinib-resistant ErbB-2-positive BC and GC.

Stat3 regulates ErbB-2 expression and co-opts ErbB-2 nuclear function to induce miR-21 expression, PDCD4 downregulation and breast cancer metastasis.

Membrane overexpression of the receptor tyrosine kinase ErbB-2 (MErbB-2) accounts for a clinically aggressive breast cancer (BC) subtype (ErbB-2-positive) with increased incidence of metastases. We and others demonstrated that nuclear ErbB-2 (NErbB-2) also plays a key role in BC and is a poor prognostic factor in ErbB-2-positive tumors. The signal transducer and activator of transcription 3 (Stat3), another player in BC, has been recognized as a downstream mediator of MErbB-2 action in BC metastasis. Here, we revealed an unanticipated novel direction of the ErbB-2 and Stat3 interaction underlying BC metastasis. We found that Stat3 binds to its response elements (GAS) at the ErbB-2 promoter to upregulate ErbB-2 transcription in metastatic, ErbB-2-positive BC. We validated these results in several BC subtypes displaying metastatic and non-metastatic ability, highlighting Stat3 general role as upstream regulator of ErbB-2 expression in BC. Moreover, we showed that Stat3 co-opts NErbB-2 function by recruiting ErbB-2 as its coactivator at the GAS sites in the promoter of microRNA-21 (miR-21), a metastasis-promoting microRNA (miRNA). Using an ErbB-2 nuclear localization domain mutant and a constitutively activated ErbB-2 variant, we found that NErbB-2 role as a Stat3 coactivator and also its direct role as transcription factor upregulate miR-21 in BC. This reveals a novel function of NErbB-2 as a regulator of miRNAs expression. Increased levels of miR-21, in turn, downregulate the expression of the metastasis-suppressor protein programmed cell death 4 (PDCD4), a validated miR-21 target. Using an in vivo model of metastatic ErbB-2-postive BC, in which we silenced Stat3 and reconstituted ErbB-2 or miR-21 expression, we showed that both are downstream mediators of Stat3-driven metastasis. Supporting the clinical relevance of our results, we found an inverse correlation between ErbB-2/Stat3 nuclear co-expression and PDCD4 expression in ErbB-2-positive primary invasive BCs. Our findings identify Stat3 and NErbB-2 as novel therapeutic targets to inhibit ErbB-2-positive BC metastasis.

Targeting ErbB-2 nuclear localization and function inhibits breast cancer growth and overcomes trastuzumab resistance.

Membrane overexpression of ErbB-2/HER2 receptor tyrosine kinase (membrane ErbB-2 (MErbB-2)) has a critical role in breast cancer (BC). We and others have also shown the role of nuclear ErbB-2 (NErbB-2) in BC, whose presence we identified as a poor prognostic factor in MErbB-2-positive tumors. Current anti-ErbB-2 therapies, as with the antibody trastuzumab (Ttzm), target only MErbB-2. Here, we found that blockade of NErbB-2 action abrogates growth of BC cells, sensitive and resistant to Ttzm, in a scenario in which ErbB-2, ErbB-3 and Akt are phosphorylated, and ErbB-2/ErbB-3 dimers are formed. Also, inhibition of NErbB-2 presence suppresses growth of a preclinical BC model resistant to Ttzm. We showed that at the cyclin D1 promoter, ErbB-2 assembles a transcriptional complex with Stat3 (signal transducer and activator of transcription 3) and ErbB-3, another member of the ErbB family, which reveals the first nuclear function of ErbB-2/ErbB-3 dimer. We identified NErbB-2 as the major proliferation driver in Ttzm-resistant BC, and demonstrated that Ttzm inability to disrupt the Stat3/ErbB-2/ErbB-3 complex underlies its failure to inhibit growth. Furthermore, our results in the clinic revealed that nuclear interaction between ErbB-2 and Stat3 correlates with poor overall survival in primary breast tumors. Our findings challenge the paradigm of anti-ErbB-2 drug design and highlight NErbB-2 as a novel target to overcome Ttzm resistance.

Ros production by endogenously generated Protoporphyrin IX in murine leukemia cells.

Endogenous production of Protoporphyrin IX (PpIX) is successfully exploited for photodynamic therapy (PDT) on malignant cells, following 5-aminolevulinic acid (ALA) administration and light irradiation. This treatment kills cancer cells by damaging organelles and impairing metabolic pathways via cellular reactive oxygen species (ROS) generation. We studied the efficiency of PpIX synthetized from ALA on ROS generation, in the Vincristine resistant (LBR-V160), Doxorubicin resistant (LBR-D160) and sensitive (LBR-) murine leukemia cell lines. Cells were incubated 4 hr with 1 mM ALA and then irradiated during different times with fluorescent light. One hour later, production of ROS was analyzed by flow cytometry using different fluorescent probes: Hydroethidine (HE) for superoxide anion, 2',7' Dichlorodihydrofluorescein diacetate (DCFH-DA) for hydrogen peroxide; mitochondrial damage was examined with 3,3' Dihexyloxacarbocyanine iodide (DiOC6). We found that superoxide anion production in the three cell lines increased with irradiation time whereas no peroxide hydrogen was detected. Mitochondrial damage also increased in an irradiation time dependent manner, being higher in the Vincristine resistant line. Previous studies have demonstrated that apoptotic cell death increased with irradiation time, which is consistent with these results, indicating that ROS are critical in ALA-PDT efficiency to kill malignant cells.

Murine abortion is associated with enhanced hyaluronan expression and abnormal localization at the fetomaternal interface.

The remodelling of the endometrial architecture is fundamental to create a suitable environment for the establishment of pregnancy. During this process, substantial alterations in the composition of maternal extracellular matrix play an important role by providing a prosperous medium for implantation as well as modulating trophoblast invasion leading to the formation of a functional placental unit. Hyaluronan is a conspicuous component of the extracellular matrix, particularly in remodelling tissues undergoing regeneration and repair. During gestation, changes in HA deposition and distribution indicate that this molecule may participate in preparation of the endometrial stroma for reception and implantation of the embryo. However, little is known about the role of hyaluronan at the fetomaternal interface, specially regarding its influence in pregnancy outcome. In the present study we show increased decidual hyaluronan levels in spontaneous abortion compared with normal pregnancy mice on gestation day 7.5. Both in normal and pathologic pregnancies, high molecular size hyaluronan was found at the fetomaternal unit. However, hyaluronan metabolism (which results from the activity of hyaluronan synthases and hyaluronidases) seems to be altered in spontaneous abortion as shown by a decrease in Hyal-3 expression as well as by differences in hyaluronan molecular size spectrum. This alteration in hyaluronan metabolism in spontaneous abortion could explain its increased concentration observed in decidua and the abnormal distribution of hyaluronan around the embryo implantation crypt. Thus, increased decidual hyaluronan levels resulting from abnormal deposition and turn over may contribute to the pathogenesis of pregnancy failure.