HER3 V104 mutations regulate cell signaling, growth, and drug sensitivity in cancer.

HER3 is mutated in ~2%-10% of cancers depending on the cancer type. We found the HER3-V104L mutation to be activating from patient-derived mutations introduced via lentiviral transduction in HER3KO HER2 + HCC1569 breast cancer cells in which endogenous HER3 was eliminated by CRISPR/Cas9. Cells expressing HER3-V104L showed higher p-HER3 and p-ERK1/2 expression versus cells expressing wild-type HER3 or HER3-V104M. Patients whose tumor expressed the HER3 V104L variant had a reduced probability of overall survival compared to patients lacking a HER3 mutation whereas we did not find a statistically significant difference in overall survival of various cancer patients with the HER3 V104M mutation. Our data showed that HER2 inhibitors suppressed cell growth of HCC1569HER3KO cells stably expressing the HER3-V104L mutation. Cancer cell lines (SNU407, UC15 and DV90) with endogenous HER3-V104M mutation showed reduced cell proliferation and p-HER2/p-ERK1/2 expression with HER2 inhibitor treatment. Knock down of HER3 abrogated cell proliferation in the above cell lines which were overall more sensitive to the ERK inhibitor SCH779284 versus PI3K inhibitors. HER3-V104L mutation stabilized HER3 protein expression in COS7 and SNUC5 cells. COS7 cells transiently transfected with the HER3-V104L mutation in the presence of HER binding partners showed higher expression of p-HER3, p-ERK1/2 versus HER3-WT in a NRG-independent manner without any change in AKT signaling. Overall, this study shows the clinical relevance of the HER3 V104L and the V104M mutations and its response to HER2, PI3K and ERK inhibitors.

HER2 inhibition increases non-muscle myosin IIA to promote tumorigenesis in HER2+ breast cancers.

HER2 is over-expressed in around 15% to 20% of breast cancers. HER3 plays a critical role in HER2 mediated tumorigenesis. Increased HER3 transcription and protein levels occur upon inhibition of HER2. We aimed to identify proteins that bound to HER3 upon inhibition of the HER family with the pan-HER inhibitor neratinib in HER2+ breast cancer cells. Immunoprecipitation of HER3 followed by mass spectrometry experiments found non-muscle myosin IIA (NMIIA) increased upon neratinib treatment relative to vehicle DMSO treatment. MYH9 is the gene that encodes for the heavy chain of NMIIA. Breast cancer patients with high MYH9 were significantly associated with a shorter disease specific survival compared to patients with low MYH9 expression from the METABRIC cohort of patients. In addition, high MYH9 expression was associated with HER2+ tumors from this cohort. Immunoblots of whole cell lysates of BT474 and MDA-MB-453 HER2+ breast cancer cells demonstrated elevated HER3 and NMIIA protein levels upon neratinib treatment for 24 hours. To examine the role of NMIIA in HER2+ breast cancer, we modulated NMIIA levels in BT474 and MDA-MB-453 cells using doxycycline inducible shRNA targeting MYH9. MYH9 knockdown reduces HER3 protein levels and concomitant reduction in downstream P-Akt. In addition, loss of MYH9 suppresses cell growth, proliferation, migration, and invasion. Our data reveals that NMIIA regulates HER3 and loss of NMIIA reduces HER2+ breast cancer growth.

HER3 Alterations in Cancer and Potential Clinical Implications.

In recent years, the third member of the HER family, kinase impaired HER3, has become a target of interest in cancer as there is accumulating evidence that HER3 plays a role in tumor growth and progression. This review focuses on HER3 activation in bladder, breast, colorectal, and lung cancer disease progression. HER3 mutations occur at a rate up to ~10% of tumors dependent on the tumor type. With patient tumors routinely sequenced for gene alterations in recent years, we have focused on HER3 mutations in bladder, breast, colon, and lung cancers particularly in response to targeted therapies and the potential to become a resistance mechanism. There are currently several HER3 targeting drugs in the pipeline, possibly improving outcomes for cancer patients with tumors containing HER3 activation and/or alterations.

IGF1R/IR Mediates Resistance to BRAF and MEK Inhibitors in BRAF-Mutant Melanoma.

The use of BRAF and MEK inhibitors for patients with BRAF-mutant melanoma is limited as patients relapse on treatment as quickly as 6 months due to acquired resistance. We generated trametinib and dabrafenib resistant melanoma (TDR) cell lines to the MEK and BRAF inhibitors, respectively. TDR cells exhibited increased viability and maintenance of downstream p-ERK and p-Akt as compared to parental cells. Receptor tyrosine kinase arrays revealed an increase in p-IGF1R and p-IR in the drug resistant cells versus drug sensitive cells. RNA-sequencing analysis identified IGF1R and INSR upregulated in resistant cell lines compared to parental cells. Analysis of TCGA PanCancer Atlas (skin cutaneous melanoma) showed that patients with a BRAF mutation and high levels of IGF1R and INSR had a worse overall survival. BMS-754807, an IGF1R/IR inhibitor, suppressed cell proliferation along with inhibition of intracellular p-Akt in TDR cells. Dual inhibition of IGF1R and INSR using siRNA reduced cell proliferation. The combination of dabrafenib, trametinib, and BMS-754807 treatment reduced in vivo xenograft tumor growth. Examining the role of IGF1R and IR in mediating resistance to BRAF and MEK inhibitors will expand possible treatment options to aid in long-term success for BRAF-mutant melanoma patients.

PI3K Inhibitors in Cancer: Clinical Implications and Adverse Effects.

The phospatidylinositol-3 kinase (PI3K) pathway is a crucial intracellular signaling pathway which is mutated or amplified in a wide variety of cancers including breast, gastric, ovarian, colorectal, prostate, glioblastoma and endometrial cancers. PI3K signaling plays an important role in cancer cell survival, angiogenesis and metastasis, making it a promising therapeutic target. There are several ongoing and completed clinical trials involving PI3K inhibitors (pan, isoform-specific and dual PI3K/mTOR) with the goal to find efficient PI3K inhibitors that could overcome resistance to current therapies. This review focuses on the current landscape of various PI3K inhibitors either as monotherapy or in combination therapies and the treatment outcomes involved in various phases of clinical trials in different cancer types. There is a discussion of the drug-related toxicities, challenges associated with these PI3K inhibitors and the adverse events leading to treatment failure. In addition, novel PI3K drugs that have potential to be translated in the clinic are highlighted.

Heteroleptic monometallic and trimetallic ruthenium(II) complexes incorporating a π-extended dipyrrin ligand: Light-activated reactions with the A549 lung cancer cell line.

A heteroleptic monometallic ruthenium(II) and a heteroleptic trimetallic ruthenium(II) complex have been synthesized and characterized. Both complexes have an overall 3+ charge, with the charge density greater for the monometallic complex. The electronic spectra of the monometallic ruthenium(II) complex exhibits intense π-π* transitions associated with the bipyridyl groups along with overlapping metal to ligand charge transfer (MLCT) and ligand centered π-π* transitions ranging from 520nm to approximately 600nm. The trimetallic ruthenium(II) complex, on the other hand, displays more well defined transitions with the expected π-π* transition of the bipyridyl groups at 294nm and Ru(dπ) to bpy(π*) MLCT transitions at 355nm and 502nm. In addition to these absorption bands an intense transition, 578nm, resulting from overlapping dipyrrin (π-π*) and Ru(dπ) to dipyrrin(π*) transitions is observed. Electrochemical and spectroelectrochemical experiments were used to help in assigning these transitions. Irradiation of the complexes in the presence of plasmid DNA within the photodynamic therapy window (600nm to 850nm) reveal, using electrophoresis, that both complexes are capable of causing photo-damage to the DNA backbone. The trimetallic ruthenium(II) complex; however, also shows the ability to generate photoinduced DNA damage in the absence of oxygen, suggesting a photo-oxidative process. Studies of the complexes toward lung cancer cells (A549 cell line) in the absence of light indicate little cytotoxicity up to 50µM. Upon irradiation of the cells with a low power 420nm light source the trimetallic complex showed considerably greater photo-cytotoxicity compared to the monometallic analog. A dose-dependent response curve gives an IC50 of 92µM for complex B.