Perseverance Is The Name Of The Game With Her3 Research In Solid Tumors
OncologyLive
Despite a reasonable expectation that expression of HER3 and its ligand heregulin would be predictive biomarkers for HER3-targeted therapy, their usefulness in clinical trials has been hit-and-miss. Nevertheless, investigators have persevered, and new types of drugs have largely supplanted the first generation in clinical trials.
Although the human epidermal growth factor receptor family of receptor tyrosine kinases plays a central role in the development of human cancers, efforts to target its activity therapeutically have focused thus far on 2 of its now famous members. Therapies aimed at the epidermal growth factor receptor and HER2, renowned as major drivers of lung and breast cancer, respectively, were among the first targeted agents in the anticancer armamentarium.
Another member of this receptor family, HER3, has also been implicated in the development of a number of cancer types but was previously largely overlooked as a therapeutic target because of its lack of kinase activity. A growing appreciation of HER3 as the principal partner for HER2 in its oncogenic activities, and accumulating evidence implicating HER3 activation as a major cause of treatment failure, drove the development of numerous HER3-targeting monoclonal antibodies during the 2000s.
A Pseudokinase
Oncogenic Double Act
Clinical Trial Failures
Table. Ongoing Clinical Development of HER3-Targeting Drugs
New Potential
Tz: Her2 Endocytosis With And Without Crosslinking Is Actin
Actin is a critical regulator of endocytosis, plasma membrane organisation and structure . Other HER2-crosslinking based approaches have suggested a role for actin/macropinocytosis in HER2 internalisation and this combined with our SEM data led us to examine the actin-dependency of crosslinking. For this we conducted control versus Tz alone versus Tz+SA experiments in the presence and absence of cytochalasin D . CytD is an actin filament disrupting agent and widely-used inhibitor of macropinocytosis . Confocal microscopy data in Figure 5A depicts SKBR3 cells incubated with Tz alone or Tz+SA in the continuous presence of CytD or DMSO diluent control. Crosslinking, as expected, increased vesicular accumulation and reduced plasma membrane localisation of Tz in control cells. However, in the presence of CytD this effect was dramatically reduced: with or without SA crosslinking Tz was largely confined to the plasma membrane. These findings were supported by Western blotting data examining levels of HER2 and P-ERK . CytD inhibited crosslinking- induced downregulation of HER2 and reduced receptor levels. Surprisingly, ERK activation was reduced by CytD treatment though the trend of Tz induced activation and enhancement by crosslinking was still evident despite being reduced in magnitude. Control data in Supplementary Figure S4A demonstrates via rhodamine-phalloidin labelling that actin is disrupted under these experimental conditions.
Her3 Mutations Are Oncogenic In Mcf10aher2 Cells And Induce Stable Heterodimer With Her2
Figure 5: Oncogenic potential of MCF10AHER2 cells expressing HER3 mutations. MCF10AHER2 cells expressing HER3EV, HER3WT and mutants were plated. Media with 2 g/ml of puromycin was replenished every second day and stained with crystal violet. The intensities are represented as mean Error bars: SEM . *P < 0.05 versus EV and **P < 0.05 versus WT. Growth kinetics of MCF10AHER2 cells with HER3 mutations was determined in vitro after 72 hr and represented in form of bar graph. Error bars: SEM, .*P < 0.01 versus EV, **P < 0.05 versus WT. Signaling in MCF10AHER2 cells expressing HER3 mutations assessed by western blot. Whole cell extracts isolated from MCF10AHER2 cells were examined using antibodies described in Materials and Methods. Actin was used as the loading control. MCF10AHER2 WT or mutants cells were immunoprecipitated using a V5 antibody. An immunoprecipitation assay was performed as described in Materials and Methods and the products were analyzed by 7.5% SDS-PAGE followed by V5, p-Tyr and HER2 immunoblots.
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N Categories For Breast Cancer
N followed by a number from 0 to 3 indicates whether the cancer has spread to lymph nodes near the breast and, if so, how many lymph nodes are involved.
Lymph node staging for breast cancer is based on how the nodes look under the microscope, and has changed as technology has gotten better. Newer methods have made it possible to find smaller and smaller groups of cancer cells, but experts haven’t been sure how much these tiny deposits of cancer cells influence outlook.
Its not yet clear how much cancer in the lymph node is needed to see a change in outlook or treatment. This is still being studied, but for now, a deposit of cancer cells must contain at least 200 cells or be at least 0.2 mm across for it to change the N stage. An area of cancer spread that is smaller than 0.2 mm doesn’t change the stage, but is recorded with abbreviations that indicate the type of special test used to find the spread.
If the area of cancer spread is at least 0.2 mm , but still not larger than 2 mm, it is called a micrometastasis . Micrometastases are counted only if there aren’t any larger areas of cancer spread. Areas of cancer spread larger than 2 mm are known to influence outlook and do change the N stage. These larger areas are sometimes called macrometastases, but are more often just called metastases.
NX: Nearby lymph nodes cannot be assessed .
N0: Cancer has not spread to nearby lymph nodes.
N1c: Both N1a and N1b apply.
N3: Any of the following:
N3a: either:
N3b: either:
Analysis Of Parp1 By Ihc
Nuclear non-cleaved PARP1 H-scores in pre-treatment FFPE resection samples were available from TNBC patients in the Nottingham cohort who ultimately received adjuvant chemotherapy, the staining and scoring of which have previously been described by Green et al.. PARP1 H-scores were compared between low/high HER3-EGFR groups by a two-tailed Mann Whitney U test using SPSS.
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Tz: Her2 Crosslinking Induces Concomitant Downregulation Of Her3 But Not Egfr
We have previously shown that SA induced crosslinking of biotinylated Tz can enhance endocytic delivery and degradation of HER2 in lysosomes . We used our previously published methods to label Tz with biotin and Alexa647, producing a fluorescent antibody amenable to crosslinking. Construct characterisation by UV spectral analysis and biotin quantification assay indicated means of 3.9 fluorophores and 6.0 biotin moieties per antibody . For functional characterisation, the Tz-construct was applied to HER2-overexpressing cells and HER2-/low cells followed by SA to induce crosslinking . Cells were imaged live by confocal microscopy at 1 and 7 hr post- crosslinking . At 1 hr, bright labelling was detected at the plasma membrane of the SKBR3 and BT474 cells with minimal evidence of binding in the MCF-7 cells indicating specificity for HER2. At 7 hr, SA-induced crosslinking lead to greater Tz internalisation in SKBR3 and BT474 cells: as demonstrated by reduced plasma membrane labelling and increased vesicular fluorescence. No differences +/- crosslinking were detected in the MCF-7 cells and collectively the results indicated the construct behaved as previously described .
Figure 1
Her3 Structure And Role In Oncogenesis
HER3, first identified by Kraus et al. is located on the long arm of chromosome 12 .9 It is encoded by 23,651 base pairs and translates into 1342 amino acids. HER3 ECD is divided into four sub-domains , which includes two cysteine-rich regions and two flanking domains that determine specificity for ligand binding. Cys-721, His-740 and Asn-815 have non-conservative substitutions in HER3 which diminishes the catalytic activity of TK domain of HER3 indicating that HER3 utilizes alternative pathway for its activation.10
Diagrammatic representation of HER2-HER3 heterodimerization, activation of downstream signaling pathways which regulate several cellular processes including cell proliferation, cell survival, apoptosis, tumor growth and metastasis.
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Her3 Ligands As Predictive Biomarkers And Anti
Several preclinical and clinical studies indicate that NRG is a predictive biomarker for HER3-targeted therapies. NRG expression correlates with in vivo tumor regression and has been validated in the clinic.151,152 In a phase I clinical trial, NRG-positive patients with metastatic or advanced tumors show a partial response to HER3-specific mAb, AV-203.153 Inhibition of HER3 using AV-203 correlates with NRG expression in preclinical studies. 117 Elevated NRG expression corroborates with HER3 activation in head and neck squamous cell carcinomas.154 However, NRG as a potential biomarker becomes questionable in tumors with HER3 mutations or downstream molecular alterations. Xenograft tumor models with a PTEN mutation show resistance to AV-203 even with elevated NRG expression.117 In HER2-amplified breast cancer model, HER3 exhibits constitutive activation independent of NRG.155
The Role Of Her3 In Resistance To Targeted Therapy
The two approaches that are frequently used in ERBB targeted therapy are: i) using monoclonal antibodies or antibody-drug conjugates or ii) tyrosine kinase inhibitors . In spite of EGFR and HER2 targeted therapies having shown tremendous success in treating a wide variety of cancers, most tumors are susceptible to resistance to these therapies within months of treatment.
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Sensitivity Of Mcf10aher2 Cells Expressing Her3 Mutants To Neratinib
Neratinib is an irreversible TKI used to treat HER2+ breast cancer patients . Bose et al. reported that a minimal concentration of neratinib is effective against HER2 mutants in MCF10AHER2 cells . Therefore, we examined whether HER3 mutants were sensitive to neratinib. Our data demonstrated that 50 nM neratinib was not significant to suppress colony formation induced by HER3F94L, HER3T355I and HER3E1261A on matrigel . A higher concentration of neratinib was effective in abrogating colony formation induced by most HER3 mutants .
Figure 7: Sensitivity of MCF10AHER2 cells expressing HER3 mutations to neratinib. MCF10AHER2 cells expressing HER3WT and HER3 mutants were seeded on a basement membrane of 3D matrigel ± 50 nM neratinib. The average size of each cellular structure was quantified. The value is represented as mean of areas ± SEM, without neratinb treatment. MCF10AHER2 cells expressing HER3WT and HER3 mutants were seeded on a basement membrane of matrigel and treated with 500 nM neratinib every second day. Phase contrast images of acini were captured at 10x magnification and the average size of each cellular structure was quantified and expressed as mean of areas ± SEM, . *P < 0.05 versus WT without neratinib treatment and **P < 0.01 versus respective HER3 mutants without neratinib treatment.
Outlook For People With Stage 3 Breast Cancer
Its natural to want to know your outlook, but statistics dont tell the whole story. Your breast cancer type, overall health, and many more factors beyond your control may affect treatment outcomes.
Establishing open communication with your treatment team can help you best assess where you are in your cancer journey.
Support groups can be a great source of comfort as you navigate your diagnosis through your treatment and beyond. Your doctors office or hospital can offer some suggestions and resources in your area.
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Abrogating Her1/her4 Erk1/2 And Er Signaling Suppresses Her3t355i Induced Proliferation In Er+ Cells
Data indicated that MAPK and HER4/HER1 pathways are activated in ER+ cells expressing HER3T355I . Lapatinib inhibits all active HER kinases . Hence, T47D and MCF-7 cells with HER3WT and HER3T355I expression were treated with lapatinib in the presence or absence of the ER inhibitor fulvestrant and the ERK1/2 inhibitor SCH772984 to analyze the effect on cell growth. The data indicated that combined treatment of lapatinib and fulvestrant reduced cell proliferation of HER3WT and HER3T355I significantly versus individual treatments in both ER+ cells. We also observed a prominent reduction in growth kinetics of these cells when subjected to co-treatment of lapatinib and SCH772984 as compared to individual agents . We performed further studies in a reconstituted basement membrane of matrigel as shown in Figure 2C and 2D. We noted significant reduction in acini formation in cells expressing HER3WT and HER3T355I when subjected to co-treatment of lapatinib and fulvestrant or lapatinib and SCH772984 as compared to individual drugs . The combined effect of 1 M lapatinib + 1 M SCH772984 or 1 M lapatinib + 1 M fulvestrant was synergistic in both ER+ cells as indicated by combination index values . Overall, these data indicate that inhibition of HER and ER signaling is sufficient to reduce the proliferation of ER+ cells expressing HER3T355I.
What Is Her2 And What Does It Mean
HER2 is a protein that helps breast cancer cells grow quickly. Breast cancer cells with higher than normal levels of HER2 are called HER2-positive. These cancers tend to grow and spread faster than breast cancers that are HER2-negative, but are much more likely to respond to treatment with drugs that target the HER2 protein.
All invasive breast cancers should be tested for HER2 either on the biopsy sample or when the tumor is removed with surgery.
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Inclusion Criteria And Category
All the articles reporting the HRs for HER3+ predicting overall survival using multivariate analysis were included in the systematic assessment. All the articles detected HER3 via IHC. The diagnostic criteria and cut-off of HER3+ expression were depicted and summarized in Table Table2.2. The diagnostic criteria were divided into three categories, and the cutoff values were divided into four categories. . HER3+ was categorized by staining intensity as 0, 1+, 2+, and 3+. 0, samples with no staining at all, or in < 10 % of the tumor cells 1+, a faint or barely perceptible incomplete staining in > 10 % of tumor cells 2+, weak-to-moderate staining in > 10 % of tumor cells 3+, strong staining in > 10-30 % of tumor cells. One cutoff value was Negative = scores 0 Positive = scores 1 + scores 2+ and 3+ another cutoff value was Negative = scores 0 and 1+ Positive = scores 2+ and 3+. . HER3+ was categorized by the percentages of positive cells × staining intensity. . HER3+ was categorized by /the numbers of tumor cores evaluated. The patients were classified into subgroup I, II, III, IV according to the diagnostic criteria and the cutoff values.
Heregulin Structure And Signaling Mechanism
A 44-kDa glycoprotein, later to be classified as an HRG, was isolated and cloned from RAS-transformed rat fibroblasts in 1992. The protein named Neu differentiation factor induced phosphorylation of p185neu and differentiation of human breast cancer cells. Almost concomitantly, another group purified and cloned a 45-kDa protein from a human breast cell line that induced phosphorylation of p185neu/HER2, which they named HRG.
Four major types of HRG proteins have been described: HRG-1 , HRG-2, HRG-3 and HRG-4. Alternate splicing of the four genes results in at least 26 different isoforms with distinct binding affinities to the HER family of receptors. The protein structure is comprised of an N-terminus motif, an Ig-like domain , an EGF-like domain, a juxtamembrane domain, a transmembrane domain, and a cytoplasmic tail. The proteins are synthesized as large membrane-anchored glycosylated precursors, with the EGFR-like motif positioned in the extracellular compartment.
Heregulins bind to HER3 or HER4 to mediate downstream signaling linked to carcinogenesisHeregulins act through paracrine, juxtacrine, or autocrine signaling
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What Should A Person With Stage 3 Breast Cancer Expect From Treatment
Stage 3 treatment options vary widely and may consist of mastectomy and radiation for local treatment and hormone therapy or chemotherapy for systemic treatment. Nearly every person with a Stage 3 diagnosis will do best with a combination of two or more treatments.
Chemotherapy is always given first with the goal to shrink the breast cancer to be smaller within the breast and within the lymph nodes that are affected. This is known as neoadjuvant chemotherapy.
Other possible treatments include biologic targeted therapy and immunotherapy. There may be various clinical trial options for interested patients with Stage 3 breast cancer.
Dual Targeting With Egfr Inhibitor And Pi3k/akt Inhibitors Reduces Nrg
Studies have shown that EGFR and HER3 upregulation and activation mediate resistance to small molecule inhibitors . We next tested the effect of NRG-1β in the presence of dual targeting with the EGFR inhibitor, gefitinib, in combination with GDC0068 or GDC0077. Tao et al. has previously demonstrated improved response rates with dual inhibition of EGFR/HER3. Figure 4A shows that NRG-1β does not significantly reduce the efficacy of gefitinib demonstrating drug dependent responses in the presence of NRG. However, combination therapy showed cell line specific responses probably linked to the expression of HER family members and/or mutational fingerprint . Combination therapy was able to reduce viability both in the absence or presence of NRG-1β in the microenvironment in the Basal TNBC cell lines but not in the Claudin type cell lines. Furthermore, combination therapy showed decreased phosphorylation of EGFR and HER3 and downstream signaling in the presence of NRG-1β in the two Basal cell lines but not the Claudin type cell line . In the MDA-MB-231 Claudin type cell line, the combination of gefitinib and AKT/PI3K inhibition had no effect with continued phosphorylation of MAPK , confirming a different reliance on EGFR family members and dominant signaling pathway. These results demonstrate differential wiring mechanisms in the two subtypes of TNBC, which should be addressed when considering the therapeutic strategy.
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Her3 Mutations Are Proliferative And Activate Mapk And Her Signaling In Er+ Breast Cancer Cells
Figure 1: Oncogenic potential of ER+ cells expressing HER3 mutations. HER3 nonsynonymous somatic mutations studied in this work depicted over HER3 protein domain. T47D cells expressing HER3EV, HER3WT and HER3 mutants were plated, treated and stained with crystal violet as described in Materials and Methods. Intensities were represented as mean Error bars: SEM ,*P < 0.05 versus EV and **P < 0.05 versus WT. MCF-7 cells with HER3 constructs were plated and stained under above similar conditions. The value is represented as mean of intensities Error bars: SEM . *P < 0.05 versus EV and **P < 0.05 versus WT. Signaling pathways in serum starved T47D and MCF-7 cells expressing HER3 EV, WT and mutants determined by western blot. Actin served as loading control. RTK arrays used to determine tyrosine phosphorylation of various RTKs from lysates of T47D and MCF-7 cells expressing HER3WT and HER3T355I serum starved overnight.